Fig. 5From: A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucoseProteasome activation is triggered by phosphorylation of Rpt6 at Serine 120. Whole cell extracts were obtained from rMC1 challenged with 25 mmol/L glucose and low glucose or mannitol for 20 and 40 min and analysed by phos-Tag assay and Wb. Phos-Tag filters were probed with antibodies anti-Rpn6, -Rpt5, -Rpt3 and -Rpt6. Rpn6 and Rpt5 blots are purposely shown overexposed to highlight the lack of species with delayed electrophoretic mobility; notably, Rpt5 migrated as a doublet band also in denaturing and reducing conditions (see Fig. 4). Wb filters were further probed with a phospho-specific antibody raised against Rpt6 phosphorylated at Serine 120 [pRpt6(Ser120] and with an anti-Rpt6 antibody. The pRpt6(Ser120)/Rpt6 ratio between immunoreactive bands was then calculated and reported in histograms. Total proteins stained by Ponceau S were used as loading control. Data are presented as mean ± SD (n = 3); a representative blot is shown. One-way ANOVA followed by Tukey’s post-hoc test: ***p < 0.001Back to article page