Polyclonal rabbit anti egfr 1005

Expression levels of EGFR, VDR and phosphorylated forms of EGFR and Akt in tissues were measured by immunoblotting of whole tongue extracts using primary antibodies specific for these markers. Western blots were performed using the following primary antibodies: polyclonal rabbit anti-EGFR (1005; Santa Cruz, Dallas, TX), monoclonal rat anti-VDR (clone: 9A7; Thermo Fisher Scientific, Waltham, MA), monoclonal rabbit anti-phospho-EGFR (phospho Y1092; abcam, Cambridge, MA), polyclonal rabbit anti-phospho-AKT1 (phospho S473; abcam, Cambridge, MA). Anti-rabbit and anti-mouse horseradish-peroxidase-conjugated secondary antibodies (GE Healthcare, Pittsburgh, PA) were used and blots were developed using enhanced chemiluminescence. Actin (I-19; Santa Cruz, Dallas, TX) was used in each experiment as a loading control. To assess difference in protein expression, western blot films were analyzed using the UN-SCAN-IT program (Silk Scientific, Inc., Orem, UT). Equal sized regions of interest (ROIs) were fitted around each expression band to measure total pixels. Using the total pixels acquired from the blot and the total pixels acquired from the corresponding actin blot, a normalized expression value was achieved [(antibody total pixels/actin total pixels)/average control tissue expression].