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Fig. 5 | Cell & Bioscience

Fig. 5

From: 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 glucose

Fig. 5

Proteasome 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 < 0.001

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