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. 2021 Jan 4;12:74. doi: 10.1038/s41467-020-20434-3

Fig. 6. Trim28 adi-KO mice demonstrate altered pathways associated with adipocyte lipid storage and metabolism.

Fig. 6

Protein analyses performed in gonadal fat pads from Trim28 wild type (WT) and adipose-specific Trim28 KO (KO) female mice fed a HFD. a Western blot from WAT of HFD-fed mice for serine 563 phosphorylation of HSL (pHSL-s563), total HSL (tHSL), total ACC (tACC), and beta-actin (β-actin). Densitometry quantification is shown for (b) HSL phosphorylation at serine 563 and (c) total ACC, (n = 6/group) *p < 0.05 versus WT mice. d Western blot from WAT of HFD-fed mice for serine 473 phosphorylation of Akt (pAkt-s473), total Akt (tAkt), serine 9 phosphorylation of GSK-3β (pGSK-3β-s9), total GSK-3β (tGSK-3β), and 14-3-3 (14-3-3). Densitometry quantification is shown for (e) Akt phosphorylation at serine 473 and (f) GSK-3β phosphorylation at serine 9, (n = 6/group for each). g mRNA expression of Park7 (DJ-1, n = 11/group), and (h) DJ-1 protein expression as determined by western blot (above, n = 6), *p < 0.05 versus WT mice. Glycerol release and HSL serine 563 phosphorylation were measured in control (shLuc) and Trim28-depleted (shTrim28) 3T3-L1 cells in response to isoproterenol treatment (0–5 µM) as a measure of lipolysis. i Glycerol release normalized to mg protein in shLuc and shTrim28 3T3-L1 adipocytes at day 8 post differentiation stimulated with 0, 0.5, 1, and 5 µM isoproterenol (n = 9/group). j HSL serine 563 phosphorylation as determined by western blot (above) normalized to total HSL and relative to shLuc vehicle (veh: 0 µM) in shTrim28 3T3-L1 adipocytes. *p < 0.05 versus shLUC, n = 3/group in triplicate. All data are presented as mean ± SEM. All data were analyzed by ANOVA with post hoc testing (Fisher’s LSD). ACC acetyl-CoA carboxylase, HSL hormone-sensitive lipase, DJ-1 protein and nucleotide deglycase DJ-1.