Fig. 5.

Activation of RSK2 by FBS or EGF stimulates endogenous RSK2 binding to LARG and LARG to RhoA. (A) U373MG cells were starved for 24 h and then stimulated with FBS (10%, 10 min) or EGF (100 ng/mL, 5 min). Endogenous RSK2 and LARG protein complex formation was determined by proximity ligation assay as described in Materials and Methods. RSK2 or LARG antibody alone was used as negative controls. Complex formation is indicated where there are distinct red spots. Complex formation in cells from each slide was quantified by ImageJ software and is presented as bar graph. (B) Starved overnight U373MG cells were treated with RSK inhibitor (RSKi, BI-D1870) for 30 min before stimulation. Complex formation is indicated where there are distinct red spots. Complex formation in cells from each slide was quantified by ImageJ software and is presented as bar graph. n = 5. (Scale bar, 5 μM.) ***P < 0.001.