Rock2

KPC or TKCC5 cells (5x10⁵) in complete media were plated into the wells of 6-well culture dishes and allowed to settle and grow overnight. Next day, cells were treated with inhibitors or DMSO vehicle in complete media for 1 h. Whole cell lysates were prepared with lysis buffer (1% SDS, 50 mM Tris pH 7.5), followed by protein concentration determination using the Bicinchoninic assay (Sigma). Standard protocols were used for immunoblotting (13 (link)). Membranes were incubated with primary antibodies: ROCK1 (BD-611136), ROCK2 (BD-610623), ROCK1/2 (Millipore 07-1458), total AKT (Cell Signaling 2920S), phospho-AKT (Cell Signaling 4060S), phospho-MYPT1 (Millipore 36003), phospho-MLC2 (Cell Signaling 3674), total MLC (MRCL3/MRLC2/MYL9; Santa Cruz sc-28329), phospho-PRAS40 (Cell Signaling 2997S), total PRAS40 (Cell Signaling 2691S), GAPDH (Millipore MAB374) and secondary antibodies: Alexa-Fluor 680 and DyLight 800 (Thermo Fisher Scientific)-conjugated antibodies. Infrared imaging (Li-Cor Odyssey) was used for detection and quantification of protein signals.