Protein Expression in Parotid Glands

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).