Coomassie plus the better bradford assay

Whole protein lysates from parotid glands of FVB, wild type C57BL/6J and Prkcz^-/- mice were harvested and processed for immunoblotting as previous described [19 (link),36 (link)]. Primary cell lysates were 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 30–100μg of total lysate was used. The following antibodies were used: anti-PARD3 (Abcam), anti-PARD6 (Proteintech), anti-total PKCζ (Cell Signaling), anti-pPKCζ (T560) (Abcam), anti-phospho-c-Jun (S63) (Cell Signaling), and anti-beta-tubulin (Thermo Scientific). Restore Western Blotting Stripping Buffer (Fisher) was used to strip membranes and reprobed for loading controls.

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.

Whole protein lysates from parotid glands of female FVB mice (n = 3/group) were harvested and processed for immunoblotting as previously described^{23 (link),86 (link)}. The Coomassie Plus-The Better Bradford Assay (Thermo) was used to determine protein concentrations and 50ug total lysate was loaded onto a 10% polyacrylamide gel, transferred to an Immobilon PDVF membrane (Millipore, Bedford, MA), and blocked in 5% nonfat milk in Tris-buffered saline-Tween 20 (1× TBST). The following antibodies were used: anti-hexokinase isoform 1 (HK1) (Product no. C35C4, Cell Signaling), anti-muscle phosphofructokinase (PFKM) (Cat. No. 55028-1-AP, Proteintech), anti-muscle pyruvate kinase isoform 1 (PKM1) (Product no. D30G6, Cell Signaling), anti-complex I—75 kDa subunit (ABN302, EMD Millipore), anti-complex III ubiquinol-cytochrome C reductase core protein 2 (UQCRC2) subunit (Cat. No. 14742-1-AP, Proteintech), and anti-extracellular signal-regulated protein kinases 1/2 (ERK1/2) (p44/42 MAPK, Cell Signaling). 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, re-block with 5% nonfat milk in 1× TBST, and re-probe for the loading control ERK1/2. Densitometry was performed using ImageJ software (NIH).