Fig 2: Increased AKT -PPP2R5D-PP2AC interaction in SNRK deficient brown and white adipose tissues. A: Co-immunoprecipitation of AKT, PPP2R5D, and PP2AC in SNRK knockout primary brown adipocytes (PBASNRK-/-) and control cells (PBAWT/WT). B–C: Co-immunoprecipitation of PPP2R5D, SNRK, and PP2AC in brown (B) and white (C) adipose tissues of SNRKloxp/loxp and SNRKA-Cre mice. D: Co-immunoprecipitation of AKT, PPP2R5D, and PP2AC in brown adipose tissue of SNRK-/-,U-Cre and littermate controls. E–F: Co-immunoprecipitation of AKT, PPP2R5D, and PP2AC in brown (E) and white (F) adipose tissues of SNRK-/-,A-Cre and littermate controls.

Fig 3: Identification of PPP2R5D as a potential novel substrate of SNRK in brown adipose tissue. A: Volcano plot representing the magnitude and significance of differential phoshpopeptide abundance in brown adipose tissue of global SNRK knockout (BATSNRK-/-) vs littermate wild type mice (BATWT/WT). B: List of phosphoproteins identified in Figures A with adjusted P value less than 0.05 and absolute value of Log2(fold change) greater than 2. C: Volcano plot representing the magnitude and significance of differential phoshpopeptide abundance in SNRK knockout primary brown adipocytes overexpressing SNRK (PBASNRK-/- + AdSNRK) vs GFP (PBASNRK-/- + AdGFP) through adenovirus-mediated infection (C) (n = 5). D: List of phosphoproteins identified in Figures C with adjusted P value less than 0.05 and absolute value of Log2(fold change) greater than 2. The overlapped phosphorylation site, PPP2R5D S80, in panels B and D, was identified as the potential substrate of SNRK in brown adipose tissue. E: In vitro validation of PPP2R5D S80 as a direct SNRK substrate. The in vitro phosphorylation reaction was conducted using purified recombinant human PPP2R5D protein in the absence/presence of active SNRK protein. The human PPP2R5D S88 is equivalent to mouse PPP2R5D S80. *P < 0.05 and **P < 0.01 as indicated. ABS, absolute value; FC, fold change; Ad, adenovirus.