THOC5 exons 4/5 were deleted from BM, colon, kidney, and liver after tamoxifen treatment. (A) The genomic structure of the exon 2 (E2) to exon 6 (E6) THOC5 locus is depicted. THOC5 deficient mice were generated as described previously . The loxP sites are located before E4 and after E5. Rosa26-CreERT2 (wild type (wt)) (n = 12) and Rosa26-CreERT2:THOC5 (flox/flox) (flox/flox) (n = 12) mice were injected with tamoxifen i. p. 2 times at 3-day intervals and were sacrificed 0, 2, 4 and 6 days after the first tamoxifen injection (3 mice for each preparation). Genomic DNA, protein and RNA were isolated from the same organs. (B) Genomic DNAs were supplied for the determination of deletion of exons 4/5 by PCR. PCR product: wild type allele: 313 bp; THOC5 E4/E5 del allele: 0 bp. As control, E20 specific primers (product: 191 bp) were supplied. (C) Protein was extracted from organs as indicated and subjected to THOC5 and actin specific immunoblot. Total protein amount was standardized by actin specific immunoblot. We performed 3 independent experiments and we show one example of representative data. (D) THOC5 and actin specific bands were quantified using the TINA 2.0 software (see Methods-Immunoblot Procedures). The signal intensity of THOC5 was standardized by actin band of each sample (3 mice for each preparation). The reduced percent signal intensity from mice treated with tamoxifen are shown. Mean values from three independent experiments are shown. The error bars represent standard deviation. (E) RNAs were extracted from liver or spleen of same mice as described above, and supplied for THOC5 (wt: 643 bp or delta E4/E5:429 bp) and GAPDH (123 bp) specific RT-PCR.
Saran et al. BMC Cell Biology 2013 14:51 doi:10.1186/1471-2121-14-51