Western Blot Analysis of Parotid Gland Proteins

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.