Figure 6: SENP1 deletion augments NEMO SUMOylation and cytokine expression in the adipocytes. | Nature Communications

Figure 6: SENP1 deletion augments NEMO SUMOylation and cytokine expression in the adipocytes.

From: SENP1-mediated NEMO deSUMOylation in adipocytes limits inflammatory responses and type-1 diabetes progression

Figure 6

(a,b). SUMOylation of NEMO, but not p65/RelA, was enhanced in the adipocytes of SENP1-aP2KO mice. Proteins extracted from the adipocytes of Ctrl and SENP1-aP2KO mice at the age of 5 weeks were subjected to immunoprecipitation with p65/RelA (a) or NEMO (b) antibodies followed by western blotting with anti-SUMO1, anti-p65/RelA or anti-NEMO. Proteins are indicated. Representative blots from one pair of Ctrl and SENP1-aP2KO mice are shown. Similar results were obtained from additional two pairs of mice. (c) Flag-tagged NEMO was transfected into adipocytes from Ctrl and SENP1-aP2KO mice at the age of 5 weeks. Proteins extracted were subjected to immunoprecipitation with SUMO1 antibody followed by western blotting with anti-flag. Input for flag-NEMO was detected with flag antibody. Representative blots from one pair of Ctrl and SENP1-aP2KO mice are shown. Similar results were obtained from additional two pairs of mice. (d) Effect of SENP1 deletion on stress-induced IKK activation, p65/RelA phosphorylation and NEMO SUMOylation. Adipocytes from Ctrl and SENP1-aP2KO mice were treated with VP16 (10 μM) for indicated times. IKK-NF-κB p65/RelA signalling molecules were determined by western blot. Ratios of p-IKK/IKK, p-p65/RelA/p65/RelA and pIκB-α were quantified by taking Ctrl as 1.0. Representative blots from one pair of Ctrl and SENP1-aP2KO mice are shown. Similar results were obtained from additional two pairs of mice. (e) VP16-induced NEMO SUMOylation was determined by co-immunoprecipitation assay with anti-NEMO (a rabbit polyclonal IgG) followed by immunoblotting with anti-SUMO1 (a rabbit polyclonal) and anti-NEMO (a goat polyclonal IgG). Representative blots from three independent experiments are shown. (f) Primary adipocytes isolated from the adipose tissue of SENP1-aP2KO mice at the age of 5 weeks were reconstituted with Flag-tagged NEMO-WT, K277R, K309R or K277/309R (DM) as detected by immunoblotting. Phosphor-p65 was quantified by taking Ctrl as 1.0. (g,h) Effect of NEMO mutants on phosphor-p65 in adipocyte was detected by intracellular staining with anti-p-p65 and FITC-conjugated secondary antibody followed by FACS with isotype IgG as a control. Representative FACS results are shown in g with quantifications in h from three independent experiments. (i) Effect of NEMO mutants on cytokine expression. Protein levels of IL-6, TNF-α, IFN-γ and IL-1β levels present in adipocyte cultures were detected with ELISA after 24-h culture. All data are means±s.e.m. of three independent experiments. The two-tailed Student’s paired t-test was used for the statistical analysis. *P<0.05; **P<0.01; ***P<0.001; Ctrl, control; IB, immunoblotting; IP, immunoprecipitation; KO, knockout.

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