PRDM16 physically interacts with HDAC3 in adipocytes, and RGFP-induced thermogenesis in primary adipocytes is blunted in the absence of PRDM16. (A) Coimmunoprecipitation of PRDM16 with HDAC3. Immortalized brown adipocytes were infected with adenovirus overexpressing GFP, FLAG-tagged HDAC3, or FLAG-tagged HDAC3 and PRDM16 as indicated. Total lysates were subjected to IP with beads specific for FLAG tag or IgG as a control. Both input and precipitates were analyzed by immunoblotting as indicated. (B–E) Real-time qPCR analyses of Prdm16 and thermogenic markers after treatment with vehicle control or 50 μM RGFP for 6 h in (B) primary brown adipocytes infected with adenovirus expressing either short hairpin RNA targeting Prdm16 (shPRDM16) or a scramble control (shSCR) (n = 3), (C) in primary brown adipocytes isolated from PRDM16f/f mice or PRDM16f/fMyf5cre mice (n = 4), (D) in primary brown adipocytes isolated from PRDM16f/f mice or PRDM16f/fAQcre mice (n = 3 to 4), or (E) in primary inguinal adipocytes isolated from PRDM16f/f mice or PRDM16f/fAQcre mice (n = 3 to 4). Data are presented as mean ± SEM; a different letter denotes a significant difference between groups at P < 0.05; ***P < 0.001. Ctrl, control; E, empty lane.