Gapdh

The OD of X-ray film protein bands was measured by densitometric quantification with LabWorks 4.0 software, adjusted and normalized to GAPDH or total corresponding proteins (UVP, Inc, Cambridge, United Kingdom). Data was expressed as %age for the maximum amount of molecule detected in each experiment. Statistical differences between groups were performed by one-way analysis of variance (ANOVA) or un-paired ‘t’ test using the GraphPad PRISM statistic software (version 7.0) (GraphPad Software, La Jolla, California USA). Statistically significant P-values (< 0.05) were considered.

Cell lysates were harvested and protein concentrations were determined via bicinchoninic acid protein quantification method [24 (link)]. 40 μg of total protein were separated by SDS-PAGE gel electrophoresis and electrotransferred onto PVDF membranes. Primary rabbit antibodies against total ERK (No. 4695), p-ERK (No. 4370), total AKT (No. 4685), p-AKT (No. 4060), KRAS (No. 3339) (Cell Signaling Technology Inc., Berkeley, CA, USA) and GAPDH (No. TA-08) (loading control) (Zhongshanjinqiao Biotech, China) were incubated at 4 °C overnight at a dilution of 1:1000, and after washed with PBST for three times, the secondary horseradish-peroxidase-labeled antibody was incubated at room temperature for 2 h at a dilution of 1:5000. Finally, the relevant protein was visualized by staining with the enhanced chemiluminescent (ECL) kit (Haigene, Harbin, China). The relative levels of each target protein to the control (total AKT, total ERK, or GAPDH) were determined using a UVP bioimaging system and LabWorks 4.6 software (UVP, Upland, CA, USA).