Fig. 2: PEX2 modulates ATGL protein levels via K48-linkage poly-ubiquitination.
From: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis
a, Co-IP conducted in HEK293T whole cell extract (WCE) via FLAG antibody 48 h after expressing PEX2–FLAG. Co-IP was analysed by IB using the indicated antibodies. Experiments were repeated three times. b, Representative images of wild-type ATGL–EGFP, ATGLΔHD–EGFP and ATGL3KR–EGFP distribution in HEK293T cells treated with 400 μM oleic acid (OA). LDs were stained by LipidTOX Deep Red dye and nuclei were stained by 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 μm. Experiments were repeated three times. c,d, IB of ectopically expressed ATGL3KR–EGFP and ATGLΔHD–EGFP in HEK293T cells 48 h after PEX2 siRNA transfection (n = 8 in PEX2 and 16 in Nc siRNA, F = 3.644 (c); n = 4 in PEX2 and 8 in Nc siRNA, F = 52.67 (d)). e, HEK293T cells were co-transfected by plasmids to express ATGL–FLAG and HA–Ub, followed by siRNA transfection. After 48 h, IP and IB were conducted to detect the ubiquitination pattern. Experiments were repeated three times. f, HEK293T cells were transfected by the ATGL–FLAG plasmid, followed by IP to enrich ATGL–FLAG for poly-ubiquitination type analysis via K48- or K63-linkage poly-ubiquitination antibodies. Experiments were repeated three times. g,h, HEK293T cells were transfected by ATGL–FLAG (g) and ATGLK92–FLAG (h) plasmids, followed by siRNA transfection. After 48 h, IP was conducted to enrich ATGL–FLAG or ATGLK92–FLAG for K48-linkage poly-ubiquitination pattern detection. Experiments were repeated three times. Results are shown as the mean ± s.e.m. and analysed using ANOVA with Dunnett correction for multiple comparisons between control and other groups.
