Figure 4. Levels of cytochrome C increased in insulin receptor substrate (IRS)-1 knockdown. 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. Mean densitometry displayed a significant increase in cytochrome c levels cultured in 25 mM glucose versus 5 mM glucose samples. Knockdown of IRS-1 protein significantly increased cytochrome C levels versus 5 mM and 25 mM glucose samples. Western blot data showing that treatment with salmeterol significantly decreased levels of proapoptotic cytochrome C (*p<0.05 versus 5 mM glucose [NT], $p<0.05 versus salmeterol n=4 for western blot).