Fig. 1: CUL2 controls PRDM16 protein stability and beige fat biogenesis.

a, Immunoblotting of Flag-tagged PRDM16 protein in HEK293T cells co-expressing Myc-tagged cullin proteins or an empty vector (Vec). b, Immunoblotting of endogenous PRDM16 protein in inguinal adipocytes overexpressing (OE) Flag-tagged CUL2 or Vec. β-Actin was used as the loading control. c, Changes in endogenous PRDM16 stability in inguinal adipocytes expressing a scrambled control shRNA (control) or shRNAs targeting Cul2 (1 and 2). Immunoblotting data are provided in Extended Data Fig. 1k. n = 3 per group. CHX, cycloheximide. d, Heat map of the RNA-seq transcriptome in differentiated inguinal adipocytes expressing a scrambled control (control) or shRNA targeting Cul2 in the presence or absence of forskolin (+cAMP). n = 3 per group. All of the listed genes are significantly different (false-discovery rate (FDR) < 0.05) by edgeR. e, The OCR in differentiated inguinal adipocytes expressing a scrambled control shRNA or shCul2. OCR values were normalized by total protein (μg). n = 7 (control) and n = 6 (Cul2 knockdown). AA, antimycin A; Nor., noradrenaline. f, The OCR in inguinal adipocytes expressing an empty vector or Cul2 normalized by total protein (μg). n = 9 for both groups. g, The OCR in inguinal adipocytes expressing a scrambled control shRNA or shCul2 from WT and Prdm16-KO mice. n = 8 (control × WT), n = 7 (shCul2 × WT), n = 9 (control × Prdm16 KO) and n = 9 (shCul2 × Prdm16 KO). For a and b, representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. 1. For b–g, data are from biologically independent samples. For c and e–g, data are mean ± s.e.m. Two-sided P values were calculated using two-way analysis of variance (ANOVA) (c) or two-way repeated-measures ANOVA (e and f) followed by Tukey test (g). *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Exact P values are shown in Supplementary Table 2.

Source data