Myosin light chain 2

Cells were washed twice in chilled 1X PBS and lysed in chilled 1X RIPA buffer supplemented with protease (Roche, Cat No. 04 693 116 001) and phosphatase inhibitors (Roche, Cat No. 04 906 837 001). Protein concentrations were determined using Pierce BCA assay kit. Samples were run on 4-20% Mini-PROTEAN pre-cast gels (Biorad, Cat No. 4568094) and transferred to PVDF membranes. The RhoActivity assay was performed using the RhoA pull-down activation assay biochem kit (Bead pull-down format) (Cytoskeleton, Cat No. BK036). The assay was carried out by incubating 800 μg of total cell lysate with 50 μg of Rhotekin as described in the manufacturers protocol following which western blots were performed. Antibodies used and dilutions are as follows: RhoA 1:1000 (Cell Signaling Technology, 2117), β-actin 1:5000 (Cell Signaling Technology, 3700S), Myosin Light Chain 2 1:1000(Cell Signaling Technology, 3672), Thr18/Ser19 Phospho-Myosin Light Chain 2 1:1000(Cell Signaling Technology, 3674), GLUT1 1:1000 (Cell Signaling Technology,12939S), GLUT4 1:1000 (Abcam, ab33780), Insulin receptor substrate 1 1:1000(Cell Signaling Technology, 2383L), Ser636/639 Phospho-Insulin receptor substrate 1 (Cell Signaling Technology, 2388S). The western blot gels were quantified by evaluating the band intensities using ImageJ (Wang et al., 2017 ).

Antibodies (and their source) against the following targets were used: fibronectin (BD Biosciences, #610077); paxillin (Abcam, ab32084); phospho-myosin light chain 2 (pThr18/pSer19; Cell Signaling Technology, #3674); myosin light chain 2 (Cell Signaling Technology, #3672); vinculin (hVin-1; Millipore-Sigma V9131); phospho-FAK (pTyr397; Cell Signaling Technology, #3283); FAK (C-20; Santa Cruz Biotechnology; sc-558); YAP1 (D8H1X; Cell Signaling Technology, #14074). DAPI (4',6-diamidino-2-phenylindole dihydrochloride), Hoechst 33342, and phalloidins conjugated to Alexa Fluor 488, 594, or 647 were from ThermoFisher Scientific. Fasudil (also known as HA-1077) was from Cayman Chemical, PF-562271 was from ChemScene, and Blebbistatin (#203389) and latrunculin A (L5163) were from Millipore Sigma. Acrylamide and N,N-methylenebisAcrylamide were purchased from National Diagnostics. Human plasma-derived fibronectin (Corning #356008) was from Thermo Fisher. Glass-bottom imaging dishes were from Cellvis. Other chemicals and reagents, unless otherwise noted, were purchased from Millipore Sigma.