Figure 4. eNOS directly interacts with PKCzeta, with both regulated by IGFBP-3. REC were transfected with IGFBP-3 NB, followed by recombinant eNOS only or eNOS and NAC in NG or HG. A: Immunoprecipitation results with eNOS beads, followed by western blotting for phosphorylated and total eNOS to demonstrate that eNOS treatment was effective. IGFBP-3 is not as effective in reducing eNOS when NAC is present. B: Immunoprecipitation results with the eNOS antibody, followed by western blotting for phosphorylated to total PKCzeta, demonstrating that eNOS interacts directly with PKCzeta. IGFBP-3 actions on PKCzeta are mediated in part through reduced peroxynitrite, as NAC increased PKCzeta compared to eNOS and IGFBP-3 only. C: ELISA for 3-nitrotyrosine on samples’ immunoprecipitated results with the eNOS antibody to demonstrate that IGFBP-3 reduces eNOS, likely through reduced peroxynitrate actions. *p<0.05 versus NG, # p<0.05 versus HG+ IGFBP-3 plasmid DNA transfection. ^$p<0.05 versus HG control DNA transfection + eNOS. % p<0.05 versus eNOS+IGFBP-3. n=3.