Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans
- Equal contributors
1 Division of Molecular Oncology, Evanston Northwestern Healthcare Research Institute, Department of Medicine, Feinberg School of Medicine, Northwestern University, Evanston, Illinois, USA
2 Division of Cancer Biology, Evanston Northwestern Healthcare Research Institute, Department of Medicine, Feinberg School of Medicine, Northwestern University, Evanston, Illinois, USA
3 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, USA
4 Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois, USA
BMC Cell Biology 2007, 8:3 doi:10.1186/1471-2121-8-3Published: 18 January 2007
Additional File 1:
Determination of the specificity of EHD peptide antisera. HEK 293FT cells in 100-mm tissue culture dishes were transiently transfected with DNA encoding a single EHD-GFP (6 μg) construct. Cell lysates were prepared as in Methods. Aliquots of 100 μg were loaded onto an 8% SDS-PAGE gel, transferred to a PVDF membrane, and immunoblotted with specific EHD anti-sera as shown. Relative molecular weight (MW) markers are indicated in kiloDaltons (kD).
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Additional File 2:
Western blot of whole cell lysates of GFP-tagged EHD proteins used in Figure 4. Aliquots of 100 μg of the lysates used for co-immunoprecipitations (co-IP) in Figure 4 were run on the same gel as those in Figure 4, transferred to PVDF membranes, and immunoblotted in parallel with anti-GFP antibodies. (A) Whole cell lysates for Figure 4A. (B) Whole cell lysates for Figure 4B. (C) Control IPs using 1 mg of whole cell lysates (WCL) and co-IPs were carried out as described in Methods using GFP-myotubularian-related protein 3 (MTMR3), Myc-sorting nexin 1 (SNX1), Myc-EHD1 and EHD1-GFP. Lanes 1–3: WCL, 100 μg. Lanes 4–6: 1 mg IP. Relative molecular weight (MW) markers are indicated in kiloDaltons (kD). The heavy chain of the mouse IgG (IgH) is also shown indicating similar levels of antibody (anti-Myc, 9e10) were used for the IP.
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Additional File 3:
Coiled-coil prediction plots of EHD proteins using COILS. Primary amino acid sequences of EHD1-4 were subjected to analysis using the COILS program  to predict the probability of the protein to adopt a coiled-coil conformation using a 28 residue scan.
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Time-lapse movie of a HeLa cell co-transfected with Rab11-GFP and EHD1-DsRed. GFP-tagged Rab11 (green) and DsRed-tagged EHD1 (red) were co-transfected into HeLa cells plated on autoclaved glass coverslips. Movie images were captured as described in Methods. Green and red vesicles are seen to move towards each other and transiently merge (yellow).
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Additional File 5:
Effect of overexpression of EHD2-4 wild type and ΔEH mutants on transferrin exit from the ERC. Methodology as described in Figure 9.
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Additional File 6:
siRNA Western Blot. (A) Lysates were prepared as described in Methods and 100 μg were loaded onto a 10% SDS-PAGE gel, transferred to a PVDF membrane, and immunoblotted with specific EHD anti-sera as shown. Relative molecular weight (MW) markers are indicated in kiloDaltons (kD). (B) The percentage (%) of remaining EHD proteins after siRNA treatment was calculated by normalizing the intensity of the EHD band with respect to the loading control and comparing it with the bands in the control siRNA-treated lanes.
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Additional File 7:
List of primers used to PCR-amplify EHD genes. Sequences corresponding to the gene are in uppercase. Sequences corresponding to the Myc-tag are italicized. Restriction enzyme sites are underlined. A "CACC" sequence was included in the forward primers for TOPO-cloning into entry vectors.
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