Fig. 8: FABP5 S-glutathionylation controls macrophage inflammation by activating PPARβ/δ.

a In vitro pull-down assays. The mouse recombinant FABP5 (WT or C127S) protein with 3xFlag tag purified from E. coli treated with GSSG (1 mM) for 15 min was incubated with COS-7 lysates and precipitated with Flag-magnetic beads, respectively. Precipitates were subjected to SDS­PAGE and examined by immunoblot with anti­ PPARβ/δ antibodies. b Transactivation assays in Raw264.7 cells transfected with a PPRE-driven luciferase reporter. After nucleofection with PPRE X3-TK-luc and pRL-TK, Raw264.7 cells were serum starved for 8 h, then exposed to H₂O₂ (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was determined, n = 3 in each group, P_{(ctr vs. H2O2)} = 0.0003, P_{(ctr vs.GW0742)} < 0.0001, P_{(GW0742 vs.H2O2+GW0742)} = 0.0047. c Transactivation assays in COS-7 cells transfected with a PPRE-driven luciferase reporter. After transfection with PPRE X3-TK-luc, FABP5, and pRL-TK, cells were serum starved for 8 h, then treated with H₂O₂ (200 μM), GW0742 (1 μM), or both for 24 h, and luciferase activity was measured, n = 3 in each group, ***P < 0.0001. d ChIP analysis of FABP5 recruitment to the PPREs of PPARβ/δ target gene promoters in Raw 264.7 cells after exposure to H₂O₂ (200 μM) for 1 h. ChIP signals were quantified by qPCR, n = 3 in each group, P_{(ADRP, ctr vs. H2O2)} = 0.001, P_{(FIAF, ctr vs. H2O2)} < 0.0001. e mRNA levels of Adrp, Fiaf, and Cpt1a in RAW264.7 cells after treatment with BMS309403 (50 μM) for 2 h, stimulation with GW0742 (1 μM) for 30 min, and exposure to H₂O₂ (200 μM) for 4 h, as determined by qPCR, n = 3 in each group, P_{(Adrp, ctr vs. GW0742)} < 0.0001, P_{(Adrp, GW0742 vs. GW0742+BMS309403)} = 0.0009, P_{(Fiaf, ctr vs. GW0742)} = 0.001, P_{(Fiaf, GW0742 vs. GW0742+BMS309403)} = 0.001, P_{(Cpt1α, ctr vs. GW0742)} < 0.0001, P_{(Cpt1α, GW0742 vs. GW0742+BMS309403)} < 0.0001. f Co-IP for S-glutathionylation of FABP5 in COS-7 cells overexpressing pXJ40-3xFlag-FABP5 WT or G114R and exposed to H₂O₂ (200 μM) for 15 min (IP, GSH; IB, FABP5). Whole-cell lysates confirm the expression of FABP5 and β-actin (DTT, negative control). All samples were biologically independent and three or more independent experiments were performed. All quantitative data are shown as mean ± SEM and analyzed with a 95% confidence interval. One-way ANOVA followed by Tukey’s post-hoc test for (b–e). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.