4.4. Co-Immunoprecipitation Assay

The P-gp binding proteins were enriched by performing a co-immunoprecipitation assay as described previously [32]. In brief, HEK-293T cells were seeded in 6-cm dishes and cultured to 70%–80% confluence. The cells were transfected with control and P-gp-Flag plasmid using the Lipofectamine 2000 method. After transfection overnight, the cells were passaged into 10-cm dishes and further cultured for another 48 h. The cells were washed with PBS and lysed in 800 μL of Triton X100-based cell lysis buffer. After centrifugation of cell lysates at 12,000× g for 30 min at 4 °C, the supernatant was precleared with Protein G-linked agarose beads. The P-gp interacting proteins were enriched by co-immunoprecipitation using anti-Flag antibody-conjugated beads. The beads were washed with cell lysis buffer and boiled with 1× SDS sample buffer at 95 °C for 5 min. The immunoprecipitated proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue R-250. MCF-7/ADR cells were lysed in Triton X100-based cell lysis buffer to analyze the protein–protein interactions. The protein lysates were initially immunoprecipitated using anti-P-gp or Rack1 or Anxa2 antibodies. The immunocomplex was further analyzed by Western blot using corresponding antibodies as described in Section 2.3. Normal rabbit/mouse IgG was used as negative controls.