Fig. 4: ROS regulates PEX2 protein levels via disulfide bond-mediated stabilization.
From: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis
a,b, HEK293T cells expressing PEX2–FLAG–Myc or iBAs expressing PEX2–FLAG were collected for IP to enrich PEX2 protein. PEX2 was eluted by Laemmli buffer with or without BME and analysed through IB via the indicated antibodies. Experiments were repeated four times. c,d, PEX2–FLAG–Myc-expressing HEK293T cells and PEX2–FLAG-expressing iBAs were treated with 0.5 mM H2O2 at different time points. IP and IB were conducted to check protein levels using the indicated antibodies at the non-reducing condition. Experiments were repeated three times. e, Human PEX2 scheme illustrating the position of 14 cysteines. Cysteines 1 to 7 are outside the RING domain, whereas cysteines 8 to 14 are inside the RING domain. PEX2 also contains two transmembrane motifs (TMs). Cysteines with a strikethrough do not exist in the murine PEX2. f, HEK293T cells were transiently transfected by plasmids to overexpress different PEX2 mutants. IP and IB were conducted at the non-reducing condition. Experiments were repeated three times. g–i, PEX2 mutants were overexpressed in HEK293T cells. After 48 h, cells were collected after 0.5 mM H2O2 treatment for 2 h. PEX2 mutants were enriched through IP for IB using the indicated antibodies. Experiments were repeated four times.
