Extended Data Fig. 1. WDR6 responds to insulin stimulation via insulin receptor, related to Fig. 1.
a, A schematic diagram illustrating the strategy for constructing genetically stable HepG2 cells with endogenous expression of WDR6-FLAG fusion protein. b, PCR identification of cells in (a). c, The effects of insulin stimulation on WDR6-FLAG protein levels and the distribution of endogenous WDR6-FLAG protein were detected by western blots. LAMINB1 and GAPDH served as a loading control for nucleus and cytoplasmic fractions, respectively. d, qPCR analysis of Wdr6 mRNA levels in siRNA-mediated Insr knockdown cells with or without insulin stimulation. n = 3 independent experiments. Actb serves as a normalization control. e, Western blots of WDR6 protein level in cells described in (d). n = 3 independent experiments. β-Actin serves as a loading control. f, Western blots of WDR6-FLAG protein levels and AKT phosphorylation levels in WDR6-FLAG cells treated with S961 (the INSR inhibitor, 10 nM) for 4 h prior to treatment with insulin (100 nM, 2 h). n = 3 independent experiments. β-Actin served as a loading control. Data in (d-f) is presented as mean ± SD, determined by two-way ANOVA and Tukey’s multiple-comparisons test. * P < 0.05, ** P < 0.01.