In the course of the diagnostic work-up, a Western blot of proteins from lymphoblastoid cells of the patient 99-3 showed a normal expression of emerin. A mutation in the emerin gene was excluded by subsequent sequencing (data not shown). Screening of the LMNA gene revealed a mutation in codon 377 (CGC > CAC) replacing Arg377 by His (LMNA R377H).

In order to study the expression level of nuclear lamin in this patient, we have separated nuclear proteins from different cell lines of controls and patient 99-3 by one-dimensional SDS-PAGE and transferred them to nitrocellulose (Fig. 1). In order to ensure that the lamin proteins have been well solubilized, the different samples were resuspended twice in the SDS-PAGE sample buffer. When nuclear proteins of the control cells were probed with antibodies against lamin A/C or B2 (lanes 1–3), the lamin antibodies reacted in all samples. In contrast, we found that in lymphoblastoid (lane 4) and fibroblast cells (lane 6) of the patient, the cellular level of lamin A/C was reduced (compare lanes 4, 6 with lanes 1, 3). Surprisingly, in myoblast cells (lane 5) and muscle tissue (lane 8) the level of lamin A/C appeared normal. The emerin antibody reacted in all nuclear extracts with a band of nearly uniform intensity indicating that equivalent amounts of protein had been loaded onto the different lanes (lanes 1–8). The same blot was re-probed with anti-fibrillarin antibodies to ensure equal protein loading (lanes 1–6 insert). We further studied the three cell types by immunofluorescence microscopy. When lymphoblastoid cells with the mutation LMNA R377H were labelled with the antibodies against emerin (Fig. 2a) or lamin B2 (Fig. 2c), all nuclei revealed the typical prominent staining of the nuclear periphery. However, after incubation with the anti-lamin A/C antibody, we found a deficiency of the immunostaining in 90% of nuclei (5121 out of 5690, Fig. 2b), while control lymphoblastoid cells showed clearly positive immunostaining of the nuclear membrane with all antibodies used (Fig. 2d,2e,2f). This result is in agreement with the immunoblot data, where lymphoblastoid cells showed a clear reduction of the lamin A/C signal.

In order to address potential tissue-specific effects of the LMNA R377H mutation we studied lamin A/C levels in other cell types from the same patient. Immunofluorescence studies of emerin, lamin A/C and lamin B on fibroblast cells revealed a pattern of intense staining at the periphery of the cell nucleus after incubation with emerin or lamin B antibodies (Fig. 3a,3c) as previously described for the lymphoblastoid cells (Fig. 2a,2c). However, we observed a difference in the staining of lamin A/C. Whereas nearly all lymphoblastoid nuclei showed a reduced or absent staining (Fig. 2b) only approximately 20% of the fibroblast nuclei (614 out of 3080) were not labelled by the lamin A/C antibody (Fig. 3b). As expected, the fluorescence of control fibroblasts showed an intense staining of the nuclear envelope with all antibodies used (Fig. 3d,3e,3f).

In the immunoblot experiments we had detected a reduced amount of lamin A/C in lymphoblastoid and fibroblast cells but surprisingly not in myoblasts and mature muscle (see Fig. 1). However, by immunofluorescence microscopy of myoblasts (Fig. 4) or frozen sections of a muscle biopsy of patient 99-3 (Fig. 5), about 5% (169 out of 3290) of myoblast nuclei (Fig. 4b) and 5% (79 out of 1580) of mature muscle nuclei showed only a faint staining with lamin A/C antibodies (Fig. 5b, arrow). In contrast, we never saw a reduction of lamin A/C staining in control myoblasts (3430 nuclei) or mature muscle (1650 nuclei). Nuclear periphery staining with emerin or lamin B2 antibodies was normal (Figs. 4, 5a,5c) as was the staining of control myoblasts and muscle sections by all antibodies (Figs. 4, 5d,5e,5f). It is interesting to note that the reduced amount of lamin A/C has been observed not only in cultured cells (Figs. 2, 3, 4), but also on frozen muscle sections (Fig. 5). This indicates that the observed reduction of lamin A/C staining is not caused by our cell culture conditions. Given that only 5% of nuclei in myoblasts and mature muscle fibres showed a reduction of lamin A/C staining it is not surprising that immunoblotting failed to demonstrate a reduction of the level of lamin A/C by (Fig. 1, lane 5).

These results support the interpretation that the cellular level of lamin A/C is reduced in cells carrying a R377H mutation in the LMNA gene. For the first time, the reduction of lamin A/C could be demonstrated by two independent methods, i.e. Western blotting and immunofluorescence microscopy. Nuclear alterations in muscle cells of an AD-EDMD have been described before 32, however, the immunohistochemical reactions with anti-lamin A/C antibodies were normal in this study.

In total, we have screened lymphoblastoid cells from another 14 patients clinically diagnosed with AD-EDMD, but we detected reduced levels of lamin A/C only in patient 99-3 and one other patient with an as yet uncharacterized genetic defect (data not shown).

In order to study the stability of the mutated lamin A/C, lymphoblastoid cells of patient 99-3 were blocked in the G1 phase by incubation in medium with 10 µM lovastatin 33. The arrest in the G1 phase was checked by FACS analysis (data not shown). After G1 phase arrest it is possible to analyze the stability of proteins during a defined time period. The level of lamin A/C expression in G1-arrested patient cells was assayed on immunoblots and compared to G1 arrested control cells. In control cells, the level of lamin A/C was stable in the presence of lovastatin over an incubation period of up to 48 h. (Fig. 6A, lanes 1–3). In patient cells, the amount of lamin A was considerably reduced by incubation with lovastatin (Fig. 6A, lanes 4–6) while the amounts of lamin C and emerin appeared only slightly reduced. This reduction is not due to an inhibition of processing 34. If so, our antibody would also recognize the pre-lamin A. Similar results were obtained after incubation with the protein synthesis inhibitor cycloheximide (50 µg/ml). The amount of lamin A and C was clearly reduced in the patient sample (compare Fig. 6B lanes 3 and 4). These data, in combination with the observation of the reduced levels of lamin A by immunoblotting and immunofluorescence microscopy support the hypothesis that the mutation R377H leads to a shorter half life of lamin A.

It has been previously described that about 10% of muscle cell nuclei from AD-EDMD patients show an alteration of nuclear morphology 3235. To examine their ultrastructure more closely, nuclei of lymphoblastoid cells (875), fibroblasts (520), myoblasts (490), and mature muscle fibres (375) from patient 99-3 and controls were analysed by electron microscopy. In lymphoblastoid cells with the mutation LMNA R377H, a striking alteration of nuclear morphology was observed. While the nuclei of control lymphoblastoid cells display a normal morphology (Fig. 7a,7a'), the nuclei of the patient cells were surrounded by a continuous double membrane with numerous blebs and the peripheral heterochromatin was no longer associated with the inner nuclear membrane (Fig. 7b, arrows). Due to the chromatin condensation, the inner pore fibrils became visible (Fig 7b', arrows). Such nuclear alterations were found in about 40% of the patient's lymphoblastoid cells. However, the cells proliferated normally indicating that the observed morphological changes are not indicative of apoptosis.

In fibroblasts (Fig. 7d,7d'), myoblasts (data not shown) and muscle sections (Fig. 7f,7f') some nuclei showed nuclear membrane invaginations (Fig. 7d',7f'), while the morphology of the membrane and the nuclear pores was not disturbed (Fig. 7d',7f' arrows). Such invaginations were found in 10% of nuclei studied, but never observed in control cells (Fig. 7c',7e'). These membrane invaginations seem to be different from both the large intranuclear channel system previously reported for muscle nuclei of X-EDMD patients and the deep invaginations of the nuclear membrane observed in an AD-EDMD patient 35. In muscle sections, the peripheral chromatin appeared more condensed compared to controls (compare Fig. 7e and 7f). This chromatin condensation was found in 20% of the nuclei studied.

Since a selective retention is required for the localization of integral proteins in the inner nuclear membrane 363738, it is possible that A-type lamins are involved in this process. Therefore, we have studied the distribution of several inner nuclear membrane proteins in different cell types from patient 99-3 and in control cells by immunocytochemistry. Emerin (Figs. 2a, 3a, 4a, 5a and 2d, 3d, 4d, 5d), LAP2β (data not shown; 3940), and MAN1 (data not shown; 41) showed a normal localization at the nuclear rim in all cell types. The presence of emerin in the nuclear envelope indicates that the interaction of emerin with the lamina is not directly affected by the mutation LMNA R377H, localized in the rod domain, as has been previously shown 26. Surprisingly, immunofluorescence microscopy revealed differences in the subcellular localization of the lamin B receptor. LBR possesses 8 membrane spanning domains and is a potential binding partner for nuclear ligands like lamin B, human chromodomain protein HP1, and DNA (see review 42). In control fibroblasts and myoblasts, LBR is highly enriched at the nuclear envelope (Fig. 8a',8c'). The same holds true for lymphoblastoid cells (data not shown). In about 95% of patient cells (2560 fibroblasts, 2840 myoblasts and 4326 lymphoblastoids), a large fraction of LBR immunostaining is found in the cytoplasm with a distribution similar to ER proteins (Fig. 8b',8d'). This was verified by double immunofluorescence using ribophorin II antibodies (data not shown). After double labelling with anti lamin A/C and anti LBR in patient fibroblasts and myoblasts (Fig. 8e",8f") we could show that the absence of lamin A/C correlates with the location of LBR to the ER (Fig. 8e,8e"). However, due to the fact that in 95% of patient cells (see above) a large fraction of LBR was found in the cytoplasm but that a lamin A/C deficiency was observed only in 5 to 20% of nuclei (myoblasts and fibroblasts, respectively), our data suggest that not the absence but the structural changes in the mutated lamin are inducing the anomalous location of LBR. While the overall levels of LBR in patient and control cells were identical when compared by immunoblotting with anti LBR antibodies (data not shown), a considerable amount of LBR is no longer retained within the inner nuclear membrane, but probably diffuses to the ER via the membrane continuities at the periphery of the nuclear pore complexes 43. However, lamin B2 still concentrates within the nuclear envelope (Fig. 8 compare a,c with b,d). These results indicate that the mislocalization of LBR correlates with the expression of the mutated lamin A.

It has been reported that lamins and lamin-associated proteins bind to chromatin and that the disruption of nuclear lamin polymers inhibits RNA pol II-dependent transcription 30. Since our data indicated that the LMNA R377H mutation impairs lamina stability we examined the distribution of phosphorylated RNA pol II in our patients and control cells. Immunofluorescence with RNA pol II antibodies revealed a normal, characteristic pattern of nuclear speckles not only in control fibroblasts (Fig. 9a") and myoblasts (Fig. 10a") (2970 and 2765 cells observed respectively) but also in fibroblast cells of patient 99-3 (Fig. 9b") (3240 cells observed). However, the distribution of phosphorylated RNA pol II was altered in 75% (1981 of 2643) of myoblasts with the mutation LMNA R377H and appeared to concentrate at the poles of the nuclei (Fig. 10b",10c", arrows). After double labelling of patient myoblasts with anti lamin A/C and anti RNA pol II (V/22) we could show that the absence or reduction of lamin A/C correlates with the altered distribution of phosphorylated RNA pol II (Fig. 10e,10e"). Similar to the results with LBR (see above), the dramatic alteration of RNA pol II distribution observed in 75% of the myoblasts compared to the reduced level of lamin A/C observed in only 5% of myoblasts suggests that the relocalization of RNA pol II within nuclei of muscle cells is a direct or indirect consequence of not only the absence but also of structural changes in lamin A. Furthermore, the lamin A mutant R377H seems to influence exclusively the localization of the phosphorylated form of RNA pol II, since the distribution of the unphosphorylated, inactive form of RNA pol II was identical to that in controls (Fig. 10d", compare to a").

In this study, we describe the effects of a defective nuclear lamin A in cells derived from AD-EDMD patient 99-3. Genomic sequencing of all the LMNA gene's exons identified a mutation in exon 6, replacing arginine 377 by histidine. The R377H mutation has been reported before in a family with limb girdle muscular dystrophy 1B 7. Like a number of other mutations it is localized in the helical rod region. By Western blotting and immunofluorescence microscopy, it was possible to demonstrate for the first time that this mutation leads to a reduction of the A-type lamin level. A less intensive staining for lamin A/C had already been reported after immunolabelling of frozen muscle and heart sections of AD-EDMD patients 2444 as well as in cultured skin cells of an X-EDMD patient 45. But until now no reduction of A-type lamin was observed by protein analysis using Western blotting. The reduction of the total lamin A level is indicative of this mutation rendering lamin A less stable. In a dominant disease, 50% of normal lamin A/C may be expected from the wildtype allele while the affected allele may produce another 50% of an altered protein. Our results suggest that mutated lamin A/C is unstable and rapidly degraded. During G1 phase arrest induced by the drug lovastatin or after protein synthesis inhibition by cycloheximide, the amount of A-type lamin in cells with the mutation R377H was decreased compared to control cells. Previous studies have reported an increased solubility of lamins in skin fibroblasts from an X-EDMD patient 46. In a recent report Vigouroux and co-workers 47 showed that a LMNA R482Q/W mutation in fibroblasts from lipodystrophic patients does not affect the nuclear content in lamins, but increases their extractability. On the other hand Östlund et al. 48 showed, after pulse-chase analysis, that several other lamin A mutants were as stable as wild-type lamin A. The difference between our findings and those reported by Markiewicz et al. 46, Vigouroux et al. 47 and Östlund et al 48 could rather reflect the effect of different lamin mutations than differences between AD-EDMD and X-EDMD or lipodystrophy. All lamins, i.e. B1, B2, A and C form dimers that assemble into multimeric filaments to form the nuclear lamina (see review 3). For this process a functional rod domain is necessary 49. The point mutation R377H is localized in the rod domain and could specifically change the 3D structure of the lamin A, disrupt intermolecular interactions involved in the normal dimerisation process, and thus affect the stability of the lamin network.

Previous histological and histochemical studies described dystrophic features in muscles from an AD-EDMD patient 4435. In this study, we found that the LMNA R377H mutation has an impact not only on lamin A stability but also on nuclear architecture. In patient lymphoblastoid cells, the nuclei showed a condensation of peripheral heterochromatin. In addition, we observed a damaged nuclear membrane with numerous blebs. Abnormal nuclear morphology of muscle and skin cells were previously observed not only in X-EDMD patients 4550 but also in an AD-EDMD patient 32. It is interesting to note that damage of nuclear membranes with numerous blebs were only observed in cells of X-EDMD patients 4550. However, similar ultrastructural abnormalities have been described in fibroblasts and hepatocytes from mice lacking A-type lamins 51. Structural abnormalities were also seen in about 10 to 20% of our patient's muscle and fibroblast nuclei. However, the nuclear membrane inclusions as well as the peripheral chromatin condensation observed in our AD-EDMD patient were different from the deep invaginations of the nuclear envelope producing pseudoinclusions reported by the group of Fidzianska who also observed strong alterations in chromatin organization in an AD-EDMD patient with a LMNA R453W mutation 35. Taken together, both studies suggest that different mutations in LMNA may cause different morphological abnormalities in nuclei. It is tempting to correlate this to the wide spectrum of clinical phenotypes associated with LMNA gene mutations 52.

The abnormality of the nuclear envelope in mutated myoblasts was also reflected by the different permeabilization conditions required for the immunolabelling of control and patient myoblasts. A staining of the nuclei and nuclear envelope from patient myoblasts was obtained when cells were permeabilized for only 2 min in -20°C methanol:aceton (1:1). In contrast, control myoblasts required at least 20 min of permeabilization. This observation indicates that the nuclear envelope of the patient's cells is more permissive for antibodies probably due to reduced stability of the lamina. Indeed, it has been previously described that an intact lamina is essential for the correct and stable reassembly of membrane vesicles in forming a nuclear envelope 2829.

From published reports 323545 and our own observations it appears that the observed nuclear alterations affect a minority of nuclei and are not tissue specific. It is, therefore, hard to conceive that the structural changes alone can cause the pathology of LMNA defects. In the case of our patient this nuclear alteration is probably due to a reduced stability of A-type lamin. In our previous work, we were able to demonstrate that an intact lamina is essential for the correct and stable reassembly of an intact nuclear double membrane around the daughter nuclei after mitosis 2829.

Other authors have demonstrated that emerin is strongly associated with the nuclear lamina 5354 and the data of Vaughan et al. 27, Harborth et al. 55, Östlund et al. 48 and Raharjo et al. 56 suggest that lamin A is essential for anchorage of emerin to the inner nuclear membrane. A direct interaction between recombinant emerin and lamin A has been shown in vitro 21. However, it was also reported that an altered lamin distribution can occur without relocalization of emerin in FPLD fibroblasts carrying R482Q, R482W or R482L mutations 4757. In accordance with these data, emerin appeared correctly localized in the nuclear membrane in cells with the LMNA R377H mutation as shown by our immunolabelling experiments. In none of our experiments did the LMNA R377H mutation have an apparent influence on emerin.

In summary, the data suggest that different amino acid substitutions elicit different effects on the intermolecular interactions of A-type lamins and that their cellular level does not play a central role in retention of emerin in the nuclear membrane. Probably only a small amount of lamin A is required to immobilize emerin into the nuclear envelope. Indeed, Harborth et al. 55 were able to demonstrate that after lamin A/C silencing, emerin re-distributed to the cytoplasm.

A set of integral nuclear proteins is known to interact with the lamina. Thus, the retention of these proteins at the inner nuclear membrane could be mediated by their binding to lamins (for review see 58). For example, the lamin B receptor (LBR) and the lamina associated protein 2β (LAP2β) interact more specifically with B-type lamins 4059, whereas emerin preferentially binds to A-type lamins 212251. In this study, we present evidence for a marked change of LBR localization in cells with the LMNA R377H mutation. In control cells, LBR is concentrated within the nuclear envelope, whereas in LMNA R377H cells there is a partial loss of nuclear envelope associated LBR, with a more general cytoplasmic distribution identical to that observed for ER proteins. Presumably LBR is no longer retained in the inner nuclear membrane and diffuses into the interconnected ER membrane system 37. These results indicate that, at least in the cells with a LMNA R377H mutation, correct LBR localization is contingent upon the level of wild-type lamin A expression. However, it has been shown that treatment of HL-60 cells with retinoic acid decreased lamin A/C to negligible amounts but LBR was still localized in the inner nuclear membrane 60. In Xenopus oocytes, peripheral chromatin but no lamin is required for the retention of LBR in the inner nuclear membrane 61. Indeed, the nucleoplasmic domain of LBR binds not only to B-type lamins 6263 but also to DNA 62 and chromosome/chromatin domains 6465 besides interacting with human chromodomain proteins 6667. Our data support the hypothesis that probably an as yet unknown chromatin protein associated to A-type lamin is involved in the retention of LBR in the nuclear envelope, and that in cells containing the probably dominant LMNA R377H mutation, this peripheral chromatin protein is no longer able to bind to the mutated A-type lamin and thus fails to establish the interaction with LBR.

Several roles have been proposed for the lamins, e.g. in nuclear envelope assembly, nuclear architecture, maintenance of chromatin organisation and DNA replication 2829686970. The implication of lamins being involved in the transcription process arose from the observation that in cells with a disrupted lamin organization, RNA polymerase II dependent transcription is dramatically impaired 3057. It has also been suggested that lamin A might affect gene expression by influencing the spatial organization of chromatin 7172. Our results support such data by demonstrating an abnormal accumulation of the phosphorylated form of RNA polymerase II which concentrates at the poles of the nuclei in myoblasts containing the LMNA R377H mutation, correlating with the failure of lamin A/C to assemble correctly.

An abnormal cytoplasmic accumulation of the RNA pol II largest subunit has been previously described in sporadic inclusion-body myositis, a progressive degenerative muscle disease of older age 73. In our study, we present the first evidence for a marked difference in intranuclear organization of the phosphorylated RNA polymerase II in cells from an AD-EDMD patient. The disorganization was specifically observed in muscle cells where the pathology of EDMD predominates and where we also found nuclear membrane inclusions. This observed nuclear change is in accord with the nuclear chromatin reorganization described in AD-EDMD by Fidzianska et al. 35.

The diagnosis of the AD-EDMD affected patient 99-3 was based on clinical findings and DNA analysis. A specific mutation in the lamin A gene was identified by direct sequencing (LMNA c.1130G>A, R377H). For a histological diagnosis, a muscle biopsy was taken from the vastus lateralis 75. In accordance with the ethical regulations of the hospital, the patient gave informed consent for further studies on his tissues. Lymphoblastoid and skin fibroblast cells (SV80 fibroblasts for control) were cultured in DMEM (Dulbecco's Modified Minimum Essential Medium) with 10% FCS and 1% L-glutamine. Myoblasts were cultured in DMEM high glucose with 20% FCS and 1% L-glutamine. Cells were grown at 37°C in a 5% CO₂ incubator. Purity of myoblast cell cultures we checked by Western blot analysis with anti-desmin antibodies as a myogenic marker (data not shown).

For some experiments, the lymphoblastoid cells were incubated for 24 h and 48 h in the presence of 10 µM lovastatin (Sigma, Taufkirchen, Germany). Under this condition, the cells are arrested in the G1 phase 33. In order to initiate the cell cycle again, medium containing lovastatin was removed and the cells were then incubated in a medium containing 2 mM mevalonic acid. For inhibition of protein synthesis, lymphoblastoid cells were incubated for 24 h in the presence of 50 µg/ml cycloheximide (Sigma). After the time of incubation the cells were washed in medium without cycloheximide. Cells were then processed for gel electrophoresis and immunoblotting.

Ascites fluids of the following primary monoclonal antibodies were used: X223 specific for the vertebrate B-type lamin B2 76, R27 against human lamin A and C 77, V/22 directed against the phosphorylated CTD region of RNA polymerase II (RNA pol II) which is located within the C-terminal domain of the large subunit of RNA pol II and required for the elongation process 78 (produced in our lab) and 8WG16 which binds specifically to the CTD heptapeptide repeat when this sequence is unphosphorylated 79. Mouse monoclonal antibodies against emerin (NCL-emerin) were purchased from medac-(Hamburg, Germany), anti desmin MA were purchased from Sigma. The monoclonal antibody P2G3 was used to detect fibrillarin 80. Antibodies against the following antigens were used: Lamin B Receptor (LBR, guinea pig; 81); lamin A/C (N-18, goat, were purchased from Santa Cruz Biotechnology, Santa Cruz, USA); Ribophorin II as an endoplasmic reticulum (ER) protein marker (rabbit; provided by Dr. B. Dobberstein).

Cultured cells grown on coverslips were fixed for 10 min in -20°C methanol, transferred for 4 min to -20°C acetone and air dried. Alternatively, the cells were fixed with methanol:acetone 1:1 for 2 min at room temperature and incubated immediately with primary antibodies. Control cell culture of muscle cells were fixed for 20 min in -20°C methanol:acetone 1:1 just before incubation with the primary antibodies. Pieces of muscle were shock frozen in isopentane cooled with liquid nitrogen. 5 µm thin cryo-sections of muscle were fixed with -20°C acetone for 10 min and air dried.

Fixed cells and cryo-sections were incubated with the primary antibodies for 30 min at room temperature. Anti-lamin B and anti-emerin antibodies were diluted 1:200 in PBS; anti-LBR, anti RNA pol II and anti-ribophorin antibodies were respectively diluted 1:800, 1:300 and 1:500 in PBS. R27 monoclonal and N-18 polyclonal antibodies, both directed against lamin A/C were used undiluted and diluted 1:100 in PBS respectively. After washing with PBS the samples were incubated for another 30 min with the appropriate secondary antibody conjugated to Texas Red or FITC (Dianova, Hamburg, Germany diluted 1:75 in PBS). The samples were then counterstained with the DNA-specific fluorescent dye Hoechst 33258 (5 µg/ml), washed in PBS, air dried from ethanol and mounted in Mowiol (Hoechst, Frankfurt, Germany).

Photographs were taken with a Zeiss Axiophot equipped with epifluorescence optics and the appropriate filter sets (Carl Zeiss, Oberkochen, Germany). Alternatively, photos were taken with a CCD Pixel Fly camera (Klughammer, Markt Indersdorf, Germany) or samples were viewed with a Leica confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany).

Cells and muscle were fixed, embedded in Epon and processed for electron microscopy according to standard procedures (for details see 82).

Proteins were resolved on 12% SDS-PAGE 83. For immunoblots, proteins were electrophoretically transferred to nitrocellulose 84. The membrane was blocked with 10% non-fat dry milk in TBST (10 mM Tris-HCl, pH = 8, 0,15 M NaCl, 0,05% Tween-20) followed by antibody-incubation at room temperature for 1 hour with anti-emerin antibodies, or anti-fibrillarin antibodies, or anti-desmin antibodies or anti-LBR antibodies (diluted respectively 1:1000, 1:500 and 1:400 in the blocking solution). After several washes with TBST the nitrocellulose was incubated with the secondary peroxidase-coupled anti-mouse or anti-guinea pig antibodies (Dianova, Hamburg, Germany) at a dilution of 1:10.000 in TBST with 10% dry milk followed for 1 hour at room temperature. The blots were washed again and bound antibodies were visualized using the enhanced chemical luminescence detection system (ECL; Amersham Buchler, Braunschweig, Germany).

For some experiments the nitrocellulose was stripped of bound antibodies and reprobed as described in the manufacturers protocol (Amersham Pharmacia Biotech) with X223 (1:1000 in TBST, 1 hour incubation) R27 (undiluted, 2 hours incubation) and P2G3 (1:500 in TBST, 1 hour incubation).