Fig. 4: The Golgi resident E3 ligase complex CUL7FBXW8 polyubiquitylates ATGL for degradation.
From: Glucose controls lipolysis through Golgi PtdIns4P-mediated regulation of ATGL
a, Quantification of ATGL protein levels in HEK293T WCE in a small-scale siRNA screen via immunoblot (n = 18 or 3 independent experiments for control or other groups). b, Immunofluorescence of ATGL in HEK293-AAV cells 72 h after CUL7&FBXW8 knockdown (n = 61 cells per group). Scale bar, 5 μm. c, Immunoblot of ATGL in the WCE, Golgi and vesicle fractions extracted from HEK293T cells 72 h after CUL7&FBXW8 knockdown (n = 6 independent experiments per group). Asterisk indicates a non-specific band. d, Immunoblot of ATGL in HepG2 cells 72 h after CUL7 and FBXW8 knockdown (n = 8 or 4 independent experiments for control or other groups). e, Representative images of LDs stained by BODIPY in HepG2 cells 72 h after siRNA knockdown. Scale bar, 20 μm. Three times repeated independently with similar results. f, Immunoblot of ATGL in iBAs 72 h after Cul7 and Fbxw8 knockdown (n = 8 or 4 independent experiments for control or other groups). g, Levels of NEFAs in the starvation medium released by iBAs in the basal state 72 h after knockdown of Cul7 and Fbxw8 (n = 6 independent experiments per group). h, Co-IP conducted in HEK293T WCE by FLAG antibody 48 h after expressing ATGL–FLAG or FBXW8–FLAG. Three times repeated independently with similar results. i, K48-linkage polyubiquitylation pattern detection in HEK293T cells transfected with ATGL–FLAG, followed by siRNA transfection for 72 h and IP to enrich ATGL–FLAG. Four times repeated independently with similar results. j, Immunoblot of ATGL in HEK293T cells 6 h after glucose withdrawal in the absence of CUL7FBXW8 (n = 5 independent experiments per group). Data are presented as mean ± s.e.m. and analysed using two-tailed unpaired t-test (b and j), two-tailed paired t-test (c), one-way ANOVA method with Dunnett correction for multiple comparisons between control and other groups (a, d and f) and Kruskal–Wallis test with Dunn’s correction for multiple comparisons between control and other groups (g). Source numerical data and unprocessed blots are available in .
