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Figure 3


Fig. 3. Phosphorylation-dependent interaction of DLC1 with 14-3-3 proteins. HEK293T cells were transiently transfected with a Myc-DLC1 expression vector and treated with (A) 100 nM okadaic acid or solvent (DMSO), or (B) 100 nM PDBu for the indicated times prior to lysis. (C) Cells were incubated with 1 µM staurosporine (ST), 5 µM Gö6983, 5 µM Gö6976 or solvent (DMSO) for 90 minutes before stimulation with 1 µM PDBu for 15 minutes. (D) MDAMB231 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{tau} beads and bound proteins were separated by SDS-PAGE. DLC1 was detected with Myc-specific antibody in A-C and DLC1-specific antibody in D (top panels). The integrity of recombinant GST-14-3-3{tau} was verified by probing the membrane with GST-specific antibody (middle panels), and expression of DLC1 was verified by immunoblotting of whole-cell extracts with Myc-specific antibody in A-C and DLC1-specific antibody in D (bottom panels).





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