Fig. 6. Stable expression of DLC1 variants in MCF7 cells inhibits proliferation. (A) Stable expression of DLC1 variants in MCF7 cells was verified by immunoblotting of whole-cell extracts using a DLC1-specific antibody (top panel). Equal loading was confirmed by probing the membrane with tubulin-specific antibody (bottom panel). (B) Proliferation of MCF7 cells stably overexpressing DLC1 WT and S327/431A, respectively, and vector control cells was measured by MTT assay. Data are normalized to absorbance at day 0. (C) MCF7 DLC1 WT and S327/431A cells were left untreated or treated with 1 µM PDBu for 15 minutes prior to lysis. Whole-cell extracts were subjected to a pull-down with GST-14-3-3 beads and bound proteins were separated by SDS-PAGE. DLC1 was detected with DLC1-specific antibody (top panel). The integrity of recombinant GST-14-3-3 was verified by probing the membrane with GST-specific antibody (middle panel), and expression of DLC1 was verified by immunoblotting of whole-cell extracts with DLC1-specific antibody (bottom panel). (D) MCF7 DLC1 WT cells were left untreated or treated with 1 µM PDBu prior to lysis. 14-3-3 proteins were immunoprecipitated from whole-cell extracts with 14-3-3-specific rabbit pAb. Immune complexes were separated by SDS-PAGE. Coprecipitated DLC1 was detected by Western blotting with DLC1-specific antibody (top panel). Immunoprecipitation of 14-3-3 proteins were verified by probing the membrane with 14-3-3-specific rabbit pAb (middle panel; IgG chains are indicated with an asterisk). Expression of DLC1 was verified by immunoblotting of whole-cell extracts with DLC1-specific antibody (bottom panel).