MSP-induced RSK2 dissociation with Erk1/2 and their phosphorylation in M-RON cells. A) MSP-induced dissociation of RSK2 from Erk1/2 in intact cells: M-RON cells (3 × 106 cells/dish) were incubated in DMEM containing 1% FBS overnight and then stimulated for 30 min with MSP (2 nM), TGF-β1 (5 ng/ml), or both in the presence or absence of 5 μM of U0126. Cellular proteins (250 μg/sample) from cell lysates were subjected to immunoprecipitation with rabbit IgG antibody specific to Erk1/2. Proteins in anti-Erk1/2 immunocomplex were subjected to Western blot analysis using antibodies specific to RSK1 or RSK2. Membranes were also reprobed with IgG antibody to Erk1/2 as the loading control. B) and C) MSP-induced RSK2 phosphorylation and its correlation with Erk1/2 activation: M-RON cells (3 × 106 cells/dish) in DMEM with 1% FBS were stimulated with MSP, TGF-β1, or both for various times. Cellular proteins (50 μg/sample) from cell lysates were subjected to Western blot analysis. Phosphorylation of RSK2 and Erk1/2 was detected by individual antibodies specific to phospho-RSK2 Ser380 or Erk1/2 T202/204, respectively. RSK2 and Erk1/2 detected by their corresponding regular antibodies were used as the loading control.