Fig. 2

High glucose stimulation induces the nuclear translocation of p65-p50 regardless of IkBα phosphorylation. A Whole cell lysates were prepared from rMC1 cells challenged with 25 mmol/L glucose for 10, 20 and 40 min or from control cells and analysed by Wb. Filters were probed with antibodies anti-pIkBα(Ser32) and -IkBα. Total proteins stained by Ponceau S were used as loading control. Results of densitometric analysis are presented as Mean ± SD (n = 3); a representative blot is shown. Statistics were calculated for high glucose vs low glucose treated cells. One-way ANOVA followed by Tukey’s post-hoc test: **p < 0.01, ***p < 0.001. B Phos-tag analysis of the same samples as in A. The filter was probed with an anti-IkBα antibody. Low and high exposures of the filter are shown. C Nuclear and cytosolic extracts were isolated from rMC1 cells challenged with 25 mmol/L glucose and low glucose or mannitol for 30 min (preliminary settings showed that stimulation for 30 min provided results more robust than those observed after 20 min). Wb analyses of these fractions was performed by using antibodies anti-p65, -p50, -lamin-B2 and β-tubulin. Histograms represent the results of densitometric analysis of the immunoreactive bands. The intensity of p65 and p50 bands of high glucose treated cells was compared to that of low glucose treated cells separately for each compartment (e.g., nucleus or cytosol). D Nuclear extracts from rMC1 cells challenged with 5 mmol/L glucose, 25 mmol/L glucose or mannitol were analysed by Trans-AM immune-enzymatic assay and DNA binding by p65 was evaluated as O.D. read at 470 nm. Statistics were calculated for high glucose vs low glucose or mannitol treated cells in C and D and data are presented as Mean ± SD (n = 3); one-way ANOVA followed by Tukey’s post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001