Extended Data Fig. 6: ROS promotes PEX2 stability via disulfide bonds.
From: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis
(a) IBAs expressing PEX2-FLAG-EGFP were collected for IP and IB by indicated antibodies. Repeated 3 times. (b) Human PEX2 was overexpressed in HEK293T cells. After treatment by 10 μM MG132 for 6 h, IP and IB were conducted to analyze PEX2 oligomerization pattern by indicated antibodies. Repeated 3 times. (c) Single cysteine to glycine mutants of human PEX2 were overexpressed in HEK293T cells. IP and IB were conducted to analyze their oligomerization patterns by indicated antibodies. Repeated 2 times. (d) Single cysteine to glycine mutants of human PEX2 were overexpressed in HEK293T cells. Cells were treated with 0.5 mM H2O2 for 2 h. IP and IB were conducted to analyze PEX2-FLAG-Myc protein by indicated antibodies. Repeated 2 times. (e-f) Wild type PEX2 or PEX2 mutants (siRNA resistant) were overexpressed in HEK293T cells or co-expressed with ATGL-FLAG, followed by siRNAs transfection. IP and IB were conducted using the indicated antibodies. Repeated 3 times. (g) PEX2 and PEX2C1-7G mutant were overexpressed in HEK293T cells, followed by 0.5 mM H2O2 treatment for 2 h. IP and IB were conducted to check K48-linkage poly-ubiquitination level. Repeated 3 times. (h-i) IBAs were transfected with Cop1 siRNAs or treated by 0.5 mM H2O2 and harvested at the indicated time points. IB was conducted using the indicated antibodies (h, N = 4 in Cop1 and 10 in Nc siRNA, F = 2.544; i, N = 3 in H2O2 treatment and 9 in control, F = 0.9765). Results are shown as mean ± SEM and analyzed using an ANOVA test with Dunnett correction for multiple comparisons between control and other groups.
