Whole protein lysates from parotid glands of FVB mice were harvested and processed for immunoblotting as previously described [11 (link), 20 (link)]. Similarly, primary cell lysates were harvested and processed in the same fashion. Briefly, samples were lysed in RIPA buffer with 5mM sodium orthovanadate (Fisher Scientific, Hampton, NH), protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), and 100mM PMSF (Thermo Scientific, Waltham, MA). The Coomassie Plus-The Better Bradford Assay (Thermo) was used to determine protein concentrations and 20-100ug total lysate was used. The following antibodies were used from Cell Signaling: anti-phospho-Yap (S127), anti-Yap, anti-Taz, anti-ERK1/2, anti-Lim domain kinase 2 (LIMK2), phosphorylated MLC, anti-MLC and anti-ROCK1. The total Yap antibody recognizes a domain on the carboxy terminus of the protein (region surrounding Pro435); therefore, it detects both phosphorylated and unphosphorylated proteins. Phosphorylated LIMK2 antibody was obtained from Thermo Scientific while pROCK antibody was obtained from Abcam. For detection, ECL substrate (Thermo Scientific) or SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) was used. Restore Western Blotting Stripping Buffer (Fisher) was used to strip the membrane, reblocked with 2% BSA in 1X TBST and reprobed for loading controls.
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