Fig. 5. Binding of 14-3-3 proteins inhibits DLC1 RhoGAP activity. (A) HEK293T cells were transiently transfected with expression vectors encoding Raichu-RhoA (con) along with Flag-DLC1 WT or S327/431A, and HA-14-3-3, where indicated. The emission ratio of Raichu-RhoA was determined by measuring YFP (FRET) and CFP fluorescence (excitation 433 nm) in cell lysates. Equal expression of 14-3-3 and DLC1 proteins was verified by immunoblotting of lysates with HA- and Flag-specific antibodies, respectively (not shown). The mean of three independent experiments performed with triplicate samples is shown; error bars represent s.e.m. Results were statistically significant for the wild-type protein (two-tailed unpaired t-test, P=0.0006); no significant difference was observed for the control and the S327/431A mutant protein (NS, P>0.05). (B) HEK293 Flp-In-DLC1 cells were left untreated (–) or treated with 10 ng/ml doxycycline (+) overnight to induce GFP-DLC1 expression. Whole-cell extracts were separated by SDS-PAGE and analyzed by western blotting with GFP-specific (top panel) and RhoA-specific antibodies (bottom panel). For SRF reporter assays, Flp-In-DLC1 cells were transiently transfected with the 3DA.Luc reporter, pTK-Renilla and HA-14-3-3 or empty vector. Cells were left untreated (–DOX) or treated with 10 ng/ml doxycycline (+DOX) for 4 hours, followed by PDBu stimulation (100 nM) for additional 4 hours. Firefly luciferase activity in cell lysates was determined and normalized by Renilla luciferase activity. Fold induction after PDBu stimulation was calculated and values for uninduced cells (–DOX) were set to 100%. Expression of DLC1 and 14-3-3 proteins was verified by immunoblotting of lysates with GFP- and HA-specific antibodies, respectively (not shown). The mean of three independent experiments performed with triplicate samples is shown, error bars represent s.e.m. (C) Flp-In-DLC1 cells were left untreated or treated with 10 ng/ml doxycycline over night and photographed with a Leitz DM IRB microscope equipped with a NPLAN 10/0.25 PH1 objective (Leica) and an AxioCam MRc camera (Zeiss) (left panels). Flp-In-DLC1 cells were transiently transfected with empty vector or EE-14-3-3 expression plasmid, followed by induction of GFP-DLC1 expression with 10 ng/ml doxycycline overnight. Cells were fixed and stained with EE-specific antibody (red) (right panels). Stacks of several confocal sections are shown. Scale bars: 20 µm. (D) Flp-In-DLC1 cells were treated with 10 ng/ml doxycycline overnight, trypsinized and kept in suspension for 1 hour. Cells were then plated onto collagen-coated dishes for the indicated times and lysed. 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 GFP-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 GFP-specific antibody (bottom panel).