FIGURE 6.

Depletion of CRK1 does not affect the localization of Sec31 to the ERES and the interaction of Sec31 with Sec13. A, effect of CRK1 depletion on Sec31 localization. Sec31 and Sec13 were endogenously tagged with PTP and a triple HA epitope, respectively, in CRK1 RNAi cell line. CRK1 RNAi was then induced for 3 days, and co-immunofluorescence microscopy was then performed with anti-protein A polyclonal antibody and FITC-conjugated anti-HA monoclonal antibody. Cells were counterstained with DAPI for DNA. B, effect of CRK1 depletion on Sec31-Sec13 complex formation, as measured by co-immunoprecipitation. Sec31 and Sec13 were tagged with a PTP epitope and a triple HA epitope, respectively, in cells containing the CRK1 RNAi construct. CRK1 RNAi was induced for 3 days, and co-immunoprecipitation was carried out in non-induced control (−Tet) and CRK1 RNAi (+Tet) cells. Tet, tetracycline. Immunoprecipitation (IP) was performed by incubating the cell lysate with IgG-Sepharose beads, and the immunoprecipitated proteins were separated on SDS-PAGE and immunoblotted (IB) with anti-HA antibody and anti-protein A antibody (anti-ProtA) to detect Sec13–3HA and Sec31-PTP, respectively. As negative controls, non-induced control and CRK1 RNAi cells expressing the 3HA-tagged Sec13 were used. DIC, differential interference contrast.