Fig. 1. Interaction of DLC1 with recombinant GST-14-3-3 proteins. (A) HEK293T cells were transiently transfected with a plasmid encoding Myc-tagged DLC1. Whole-cell extracts (WCE) were incubated with glutathione beads coupled to the indicated GST–14-3-3 isoforms, or GST alone, and bound proteins were separated by SDS-PAGE. DLC1 was detected by western blotting with a Myc-specific antibody (top panel). The integrity of GST (marked with an arrowhead) and GST-14-3-3 isoforms (marked with an arrow) was verified by probing the membrane with GST-specific antibody. (B) Whole-cell extracts of HEK293T cells transiently expressing Myc-DLC1 were subjected to a pull-down (PD) with wild-type (WT) or R56/60A GST-14-3-3, and bound proteins were analyzed as described in A. (C) Schematic representation of DLC1 truncation mutants and putative PKD phosphorylation sites. (D) Whole-cell extracts of HEK293T cells transiently expressing the indicated Flag-tagged DLC1 mutants were subjected to a pull-down with GST-14-3-3β and bound proteins were analyzed with Flag-specific antibody as described in A. Expression of the different DLC1 variants was verified by immunoblotting of whole-cell extracts with Flag-specific antibody (right panel).