Fig. 2. Effect of the MEK5/ERK5 knockdown on cell proliferation of lung cancer cells.
a MEK5 mRNA expression was determined by quantitative RT-PCR in NSCLC cell lines. GAPDH was used as control for differences in cDNA input. Data show the mean expression ± SD of an experiment that was repeated twice, calculated according to the ΔΔCt relative quantitation method. b Expression of MEK5 and pMEK5 in NSCLC cell lines. 50 µg of whole-cell lysates were used to detect MEK5 by western blotting. For pMEK5 detection, 2 mg of protein were immunoprecipitated with the anti-MEK5 antibody followed by western blotting with the anti-pMEK5 antibody. GAPDH was used as loading control. c Effect of MEK5 knockdown on cell proliferation. Knockdown of MEK5 was carried out by lentiviral infection of NSCLC cells with a scrambled control sequence or MEK5 specific shRNAs (sh66 and sh68) and levels of MEK5 were evaluated by western blotting. The effect of MEK5 knockdown on cell proliferation was measured by MTT assay at the indicated times. Data are presented as the mean ± SD of an experiment that was repeated three times. p-values were calculated according to a two-sided Student’s t-test. d Expression of total ERK5 in NSCLC cell lines. pERK5 and total ERK5 were evaluated by immunoprecipitating 1 mg of protein with the anti-ERK5 antibody and blots probed with the C-terminal anti-ERK5 antibody. GAPDH was used as loading control. e pERK5 quantitation in NSCLC cells. pERK5 (upper band) and ERK5 (lower band) levels from five independent western blot studies were quantified with the ImageJ software and the pERK5/ERK5 ratio represented. f Silencing of ERK5 in NSCLC cells by shRNA was confirmed by western blotting with the anti-ERK5 antibody. GAPDH was used as loading control. g Proliferation effect of ERK5 knockdown was evaluated at 3 days as in (c).