Fig. 1

Direct interaction of HCR with astrin. A Reciprocal co-immunoprecipitation analysis of HCR binding to astrin. HeLa cell lysates were immunoprecipitated with astrin, HCR, or control rabbit IgG antibodies and analyzed by western blotting with anti-astrin and anti-HCR antibodies. Anti-GM130 and anti-beta actin antibodies were used as negative controls. B GST pull-down assay of the interaction between astrin and GST-tagged HCR. Total lysates of HeLa cells expressing GFP-astrin were incubated with GST alone or GST-HCR purified from bacterial cells. Precipitates were detected with an anti-GFP antibody. C Schematic models of the deletion mutants of HCR and astrin. D Co-immunoprecipitation analysis of the astrin-binding domain on HCR. GFP vector alone or each HCR-GFP fragment were co-transfected with myc-astrin into HeLa cells, and then, lysates were immunoprecipitated with an anti-myc antibody and analyzed by anti-myc and anti-GFP antibodies. E Co-immunoprecipitation analysis of the HCR-binding domain on astrin. PCMV-myc empty vector or each myc-astrin fragment was co-transfected with HCR-GFP into HeLa cells, and then, lysates were immunoprecipitated with an anti-myc antibody and analyzed by anti-GFP and anti-myc antibodies. F Co-immunoprecipitation analysis of the interactive domains between HCR and astrin. GFP vector alone or GFP-HCR-CC3 fragment was co-transfected with myc-astrin-CC2 into HeLa cells, and then, lysates were immunoprecipitated with an anti-myc antibody and analyzed by anti-GFP or anti-myc antibodies. G In vitro analysis of the domain in HCR required for interacting with astrin. GST-tagged astrin and His-tagged HCR fragments were purified from E. coli strain BL21(DE3), and a pull-down assay was performed to examine the astrin-binding domain in HCR