p62 expression under oxidative stress is dependent on NFκB p65 Ser536 phosphorylation.

(A) ARPE-19 cells were treated with H₂O₂ (400μM) for 3, 6 and 12 hours and p62, NFκB p65 ser 536 phosphorylation, and total NFκB p65 protein levels were measured by western blot. β-actin was used as an internal control. Representative western blot from atleast 3 independent experiments has been shown. (B) NFκB p65 nuclear translocation was determined after treating ARPE-19 cells with H₂O₂ (400μM) for 3, 6 and 12 hours and western blotting on separated nuclear and cytosolic fraction lyastes. Purity of cytosolic and nuclear fractions were determined by blotting for tubulin and Histone (H3) respectively. Representative Western blot from at least 3 independent experiments are shown. (C) p62 mRNA levels were measured by qRT-PCR after pretreating the ARPE-19 cells with IKKβ inhibitor SC-514 (100μM) for 1 hour and subsequently co-treating with H₂O₂ (400μM) for 3 or 24 hours. (D) ARPE-19 cells were pretreated with IKKβ inhibitor SC-514 for 1 hour and subsequently co-treated with H₂O₂ (400μM) for 6 or 24 hours. p62, LC3, NFκB p65 ser 536 phosphorylation, and total NFκB p65 protein levels were measured by western blot. β-actin was used as an internal control. Representative Western blot from at least 3 independent experiments is shown. (E) ARPE-19 cells were either treated alone or co-treated with competing NFκB Ser529, 536 short peptide or co-treated with H₂O₂ (400μM) for 3 hours. p62 mRNA levels were measured by qRT-PCR. (F) ARPE-19 cells were either treated alone or co-treated with competing NFκB Ser529, 536 short peptide or co-treated with H₂O₂ (400μM) for 3 or 24 hours. p62 and NFκB p65 ser 536 phosphorylation protein levels were measured by western blotting. Ctrl = Untreated control; Pep = NFκB Ser529, 536 short peptide treated. (G) Empty pcDNA3.1 vector, p65-WT, dominant negative mutant p65-S536A and dominant positive mutant S536D were transiently overexpressed in ARPE-19 cells for 24 hours and cell lysates were analyzed for NFκB p65 ser 536 phosphorylation and total NFκB p65 protein levels by western blotting. (H) ARPE-19 cells transfected with respective plasmids for 24 hours were either left untreated or treated with H₂O₂ (400μM) for an additional 3 hours and p62 mRNA levels were measured by qRT-PCR. Data represent the mean +/- S.E.M. for three samples per group. Differences in means were considered statistically significant when p<0.05. (I) p62, NFκB p65 ser 536 phosphorylation and total NFκB p65 protein levels were measured by Western blot. β-actin was used as an internal control. Representative western blot from at least 3 independent experiments is shown.