A New Model for Raf Kinase Inhibitory Protein Induced Chemotherapeutic Resistance
A, Western blotting (lower panel) and β-actin normalized densitometric measurements (upper panel) for KEAP 1 in HEK-499 and control cells. B, Western blotting and β-actin normalized densitometric measurement (bar charts) for KEAP 1 in RKIP induced cells (Doxy+/RKIP++ are Flp-In T-Rex 293 cells stimulated with Doxycycline and Doxy−/RKIP+ are not treated/stimulated). C, Western blot and densitometric measurements of KEAP 1 protein in HT29 CRC cells transfected with empty vector (EVC) or pBP vector that overexpresses flagged RKIP (fRKIP). D, Quantitative RT-PCR of normalized KEAP 1 mRNA fold changes in HEK-293 control and HEK-499 RKIP depleted cells. E, GAPDH (left) β-actin (right) normalized KEAP 1 fold changes in mRNA expression in HT29 control (KEAP1-1 pBP) and flagged RKIP (KEAP 1-1 RKIP) transfected cells. F, HEK-293 control and HEK-499 RKIP depleted cells exposed to cycloheximide (CHX; 35 µM) for the indicated times. KEAP 1 and GAPDH protein levels were analyzed by Western blotting. G, Western blotting for RKIP, KEAP 1 and tubulin in HT29 CRC cells transfected with empty vector (EVC) or pBP vector that overexpresses flagged RKIP (fRKIP) exposed to cycloheximide (CHX; 35 µM) for the indicated times. All western experiments were performed at least in duplicates and repeated twice. Asterisks indicate statistical significance (p<0.05 compared to equivalent controls).