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. 2015 Jul 31;29(12):4772–4782. doi: 10.1096/fj.15-275453

Figure 6.

Figure 6.

p62 and PKCζ association is required for hyperglycemia to stimulate NF-κB pathway-dependent proteins and for IGF-I–stimulated downstream signaling. A, D, and G) VSMCs were cultured in DMEM containing normal glucose (5 mM) plus 10% FBS then serum deprived for 16 h before treatment with 25 mM glucose (NHG) for 6 h in the presence of a control peptide (CP) or a disrupting peptide (AP) (10 μg/ml). B, C, E, and H) VSMCs expressing shRNA targeting LacZ (Ctrl Si) or p62 (p62 Si) were cultured in DMEM containing normal glucose (5 mM, NG) plus 10% FBS then serum deprived for 16 h before treatment with 25 mM glucose (NHG) for 6 h or maintained in NG. IGF-I (100 ng/ml) was added [1 min treatment for (D and (E), or 10 min treatment for (G) and (H)] before cells were harvested. A and B) Cell lysates were immunoblotted (IB) using an anti-Nox4, anti–IL-6, or anti–TNF-α antibody. To control for loading, the blots were stripped and reprobed with an anti-β-actin antibody. C) ROS was measured as described in Materials and Methods. D and E) Cell lysates were immunoblotted with an anti-p419 Src antibody. Cell lysates were also immunoprecipitated (IP) with an anti-Src antibody and immunoblotted with an anti-DNP antibody (D). To control for loading, the blots were stripped and reprobed with an anti-Src antibody. F) VSMCs were cultured in DMEM containing normal glucose (5 mM) plus 10% FBS then serum deprived for 16 h before treatment with 25 mM glucose (NHG) for 6 h. Prior to 25 mM glucose exposure, cells were preincubated with or without Bay 11-7082 (10 μM), an NF-κB inhibitor (NF-κB in), for 30 min. After 6 h high-glucose exposure, IGF-I (100 ng/ml) was added for 1 min treatment. Cell lysates were immunoblotted with an anti-pTyr419 Src antibody. To control for loading, the blots were reprobed with an anti-Src antibody. G and H) Cell lysates were immunoblotted with an anti-pAKT (Ser473) or anti-pErk1/2 antibody. To control for loading, the blots were stripped and reprobed with an anti-AKT or anti-Erk1/2 antibody. p, NS, no significant difference in P value. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences between 2 treatments.