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Fig. 6 | Cell & Bioscience

Fig. 6

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. 6Fig. 6

CamKII, but not PKA, phosphorylates Rpt6. A rMC1 cells were pre-treated with 10 µM DMSO, Inh. XII or KT2750 for 2 h. Thereafter, compounds were washed out and 25 mmol/L glucose or low glucose/mannitol was added to culture media for 20 min and crude cell extracts were prepared. Cells treated with low glucose and mannitol were used as controls. Bulk chymotrypsin like activity was determined as described in Fig. 4C and results expressed as relative activity respect to the slope of Suc-LLVY-amc cleavage by proteasome in crude cell extracts of low glucose stimulated cells. One-way ANOVA followed by Tukey’s post-hoc test (n = 3): **p < 0.01. B The same crude cell extracts were also run by native-gel electrophoresis (as in Fig. 4D) and the filters probed with the anti-Rpt5 antibody. An aliquot of the sample was run by denaturing and reducing Wb to rule out uneven gel loading (bottom panel). C, D Whole cell extracts were obtained from rMC1 challenged with 25 mmol/L for 20 and analysed by phos-Tag assay (C) and Wb (D). Phos-Tag filters were probed with the anti-Rpt6 antibody. Wb filters were probed with the phospho-specific anti-Rpt6(Ser120) antibody and with the anti-Rpt6 antibody. The pRpt6(Ser120)/Rpt6 ratio was then calculated from immunoreactive bands of high glucose vs low glucose lanes and shown in histograms. Total proteins stained by Ponceau S were used as loading control. Data are presented as mean ± SD (n = 3), a representative blot of three independent replicates is shown. One-way ANOVA followed by Tukey’s post-hoc test: ***p < 0.001

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