Co-immunoprecipitation of GEP and GRP78 Proteins

Co-immunoprecipitation was performed as described previously [20 (link)] with minor modifications. Briefly, cells were cultured in T175 flasks for 3 days under normal conditions until the cells reached around 80% confluence. The membrane fraction enriched protein lysate described in the earlier section was incubated with monoclonal antibody anti-GEP antibody A23 (Versitech) [17 (link)] or anti-GRP78 antibody (Cell Signaling), respectively, at a ratio of 400 μg to 2 μg. This mixture was incubated at 4 °C overnight under rotation. Antibody alone and protein lysate alone, respectively, were performed as independent control reactions and served as references for the non-specific bindings with Protein G Sepharose. For each reaction, 100 μl of equilibrated protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) were added to the antibody-lysate mixture and incubated at 4 °C with rotation for an hour. The complexes were briefly centrifuged after incubation. The supernatant was discarded and the beads were washed with 500 μl lysis buffer for 5 times to remove any unbound proteins. The co-immunoprecipitated proteins were eluted by adding SDS sample buffer to the protein G-Sepharose beads followed by 5 min incubation at 95 °C.