Fig. 3: SETD5 is transiently stabilized and degraded by APC/C and proteasome during the early adipogenesis. | Nature Communications

Fig. 3: SETD5 is transiently stabilized and degraded by APC/C and proteasome during the early adipogenesis.

From: Spatiotemporal dynamics of SETD5-containing NCoR–HDAC3 complex determines enhancer activation for adipogenesis

Fig. 3

a Immunoblot showing expression of endogenous SETD5 in 3T3-L1 preadipocytes. b Immunoblot showing expression of SETD5-V5, NCoR2, HDAC3, and CDC20 in SETD5-transduced 3T3-L1 preadipocytes. The graph shows the band intensity of SETD5-V5 and CDC20. c Cell cycle analysis of empty virus-transduced 3T3-L1 preadipocytes. The ratios of the cells at G1, S, or G2/M phase are shown. d Immunoblot showing expression of SETD5-V5 in SETD5-transduced 3T3-L1 preadipocytes treated with MDI and MG132. The graph shows band intensity of SETD5-V5. e Immunoblot showing ubiquitinated SETD5-V5. SETD5-transduced 3T3-L1 preadipocytes were treated with MDI and then MG132 at 9 h after MDI induction. Preadipocytes were subjected to immunoprecipitation using anti-V5 antibody and immunoblotting with anti-ubiquitin and anti-V5 antibodies. f Heatmap representing the abundance of APC/C subunits and RNF213 in SETD5-V5 immunoprecipitates. Spectrum count for each protein in proteomics analysis (Fig. 1j) was normalized by spectrum count for common region of SETD5 (a.a. 1 to a.a. 272 and a.a. 919 to a.a. 1441). g Expression of mRNAs of APC/C subunits during the early adipogenesis. mRNA expression of Cdc20 and Anapc2 in SETD5-V5-transduced preadipocytes were quantified by qPCR. h Immunoblot showing SETD5-V5 stability by the cycloheximide chase assay. 3T3-L1 preadipocytes transduced with SETD5-V5 were transfected with control siRNA or siRNA targeted to Anapc11 and induced for differentiation with MDI mixture. Preadipocytes were treated with cycloheximide (CHX) at 9 h after MDI induction and subjected to immunoblot analysis with anti-V5. Representative of three independent experiments. The graph shows regression analysis of SETD5-V5 protein stability after CHX treatment. Data are mean ± SEM of three independent experiments. Protein half-life (T1/2) was calculated based on exponential decay curve fit to the average data at each time point. a, b, d Equal loading of the protein was confirmed by blotting with anti-HDAC1 (a) or anti-actin β (b, d) antibody. a, b, d, e, g Representative of three (a, b) or two (d, e, g) independent experiments. c, g Data are mean ± SD of three technical replicates. Source data are provided as a Source data file.

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