Fig. 4: WDR6 interacts with PPP1CB to facilitate the latter’s dephosphorylation at Thr316.
From: Upregulation of WDR6 drives hepatic de novo lipogenesis in insulin resistance in mice
a, Schematic of immunoprecipitation using mass spectrometry (IP–MS) to identify proteins associated with WDR6 in WDR6–FLAG cells (left) and phosphoproteomic analysis to identify differential phosphorylated proteins in primary hepatocytes of WT and WDR6-WKO mice, respectively (right). Petri dish, mouse and microtube icons produced using Servier Medical Art by Servier (https://smart.servier.com/) under a Creative Commons Attribution 3.0 Unported Licence. Image of mass spectrometer reproduced with permission from Jingjie PTM BioLab. b, Eluted fraction from IP indicated in a was analysed by SDS–PAGE with Coomassie Brilliant blue staining (left), and top five of the WDR6-associated proteins identified in MS are listed (right). The black arrow represents the position of PPP1CB. c, A volcano plot of differential proteins shown in phosphoproteomic analysis of primary hepatocytes isolated from WT and WDR6-WKO mice. There were 19 downregulated proteins (black) and 39 upregulated proteins (red). d, Representative IP analysis of the interaction of endogenous WDR6–FLAG fusion protein with endogenous PPP1CA, PPP1CB or PPP1CC in HepG2 cells. e,f, Representative IP analysis of the interaction of WDR6–FLAG and PPP1CB-HA (e) and PPP1CB-FLAG and HA-WDR6 (f) in HEK293 cells. GAPDH served as the loading control. g,h, Western blots of phospho-PPP1CB (Thr316) in liver of WDR6-LKI (g) and WDR6-LKO (h) mice. n = 4 biologically independent mice per group. β-actin served as the loading control. i–k, Western blots of phospho-PPP1CB (Thr316) and FASN levels in liver in response to HFD-induced IR state (n = 4 biologically independent mice per group) (i), insulin stimulation (0.75 units per kg body weight, i.p.) for 6 h (n = 4 biologically independent mice per group) (j) and normal feeding, overnight fasting (n = 4 biologically independent mice) and 6 h of refeeding (n = 6 biologically independent mice) (k). β-actin served as the loading control. Experiments in c and g–k were performed using male mice. Data in g–j are presented as the mean ± s.d., determined by unpaired two-sided Student’s t-test. Data in k are presented as the mean ± s.d., determined by one-way ANOVA and Tukey’s multiple-comparisons test.
