Proteins were transferred to polyvinylidene difluoride membranes and blocked with Odyssey blocking buffer, probed with primary antibodies in blocking buffer, and detected and quantified on the Odyssey system (LI-COR). The following antibodies were used: mouse monoclonal and rabbit polyclonal anti-actin (1:5000 WB; Sigma-Aldrich), mouse monoclonal anti-RLC20 (1:1000 WB; Sigma-Aldrich), rabbit polyclonal anti-RLC20 phospho-Ser19 (1:200 WB; Cell Signaling), mouse monoclonal anti-MYPT1 (1:1000 WB; BD Biosciences), rabbit polyclonal anti-MYPT1 Thr853 (1:200 WB; Cell Signaling), rabbit polyclonal anti-MYPT1 Thr696 (1:500 WB; Cell Signaling), rabbit polyclonal anti-MYPT1 Thr668 (1:500 WB; Cell Signaling), rabbit polyclonal antibodies anti-RSK2 phospho-Ser227/phospho-Thr577 and mouse monoclonal anti-RSK2 (1:500 WB; 1:50 IP; Santa Cruz Biotechnology Inc.), phospho-Ser227/phospho-Thr577 blocking peptides SC12445 and SC3744664 (Santa Cruz Biotechnology Inc.), sheep polyclonal anti–NHE-1 phospho-Ser703 (1:500 WB; MRC-PPU Reagents), mouse monoclonal (1:200 WB; Millipore) and rabbit polyclonal anti–NHE-1 (1:100 WB; LSBio), mouse monoclonal anti-MLCK (1:500 WB; Sigma-Aldrich), mouse monoclonal anti-caveolin2 (1:200 WB; Cell Signaling), rabbit polyclonal anti-histone3 (1:200 WB; Cell Signaling), rabbit polyclonal anti-MYH11 (1:500 WB), mouse monoclonal anti-tubulin (1:5000 WB; Millipore), GAPDH (1:5000 WB; Millipore), anti-LARG (1:100 WB; Santa Cruz Biotechnology Inc.), p63RhoGEF (GEFT) (1:200 WB; Proteintech Group Inc.), anti-GEFH1 (1:200 WB; Cell Signaling), rabbit polyclonal anti-Gαq/11 (1:500 WB; Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-Gα12 (1:300 WB; Santa Cruz Biotechnology Inc.), and anti-ROCK2 goat polyclonal (1:300 WB; Santa Cruz Biotechnology Inc.). Primary antibodies were detected with either goat anti-rabbit or anti-mouse Alexa Fluor 680 (1:10,000, Invitrogen), donkey anti-sheep Alexa Fluor 680 (1:10,000, Invitrogen), or a goat anti-rabbit and anti-mouse IRDye800 (1:10,000, Rockland Immunochemicals)–conjugated secondary antibody.

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