Figure 5. Bax protein levels increased in insulin receptor substrate (IRS)-1 knockdown. Protein levels of Bax were significantly increased 25 mM glucose versus 5 mM glucose samples. Transfection of IRS-1 shRNA in Müller cells significantly increased levels of pro-apoptotic Bax. Salmeterol (10 uM) significantly decreased Bax levels activity after 6 h of treatment. Significance was determined by the Mann–Whitney test (*p<0.05 versus 25 mM glucose, $p<0.05 versus salmeterol n=4, #p<0.05 versus 5 mM glucose, n=4 for western blot). Equal loading was verified using ponceau staining. Mean densitometry was done for each blot which consisted of taking the mean optical densities of 4 different western blots for each protein analyzed.