Fig. 5: S-glutathionylation promotes the nuclear translocation and fatty acid binding of FABP5. | Nature Communications

Fig. 5: S-glutathionylation promotes the nuclear translocation and fatty acid binding of FABP5.

From: Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

Fig. 5

a–c Immunoblot analysis of FABP5 binding to ULCFA (a), SLCFA (b) or ULCFA with Grx1 treatment (c). Purified recombinant FABP5 protein was incubated with GSH plus H2O2 or GSSG for 15 min, then mixed with Biotin-ULCFA or Biotin-SLCFA in the absence or presence of BMS309403 or linoleic acid or Grx1. FABP5 interacted with the Biotin-ULCFA or Biotin-SLCFA was captured using streptavidin magnetic beads. d Immunofluorescence staining of BMDMs after exposure to H2O2 (200 μM) for 30 min (green, FABP5; blue, DAPI; scale bars, 10 μm). e Quantitative analysis of the ratio of nuclear/cytoplasmic fluorescence intensity in BMDMs as in (d), n = 10 in each group, P(ctr vs. ctr H2O2) = 0.0172, P(ctr H2O2 vs. Grx1 KO H2O2) <0.0001. f Immunoblot analysis of cytoplasmic and nuclear FABP5 in COS-7 cells. COS-7 cells were transfected with pXJ40-3xFlag vector or pXJ40-3xFlag-FABP5 followed by exposure to H2O2 (200 μM) for 1 h. Lamin B1 (nuclear fraction) and GAPDH (cytoplasmic fraction) are loading controls. g Immunoblots with antibodies against the indicated proteins in nuclear and cytoplasmic fractions from COS-7 cells co-transfected with pXJ40-3xFlag-FABP5 (or vector) and pcDNA3.1-3xFlag-Grx1 (or vector) for 24 h, then exposed to H2O2 (200 μM) for 1 h. h Immunoblots against the indicated proteins of cytoplasmic and nuclear extracts from BMDMs exposed to H2O2 (200 μM) for 1 h. i mRNA expression of Il1b, Il6, and Tnfα in BMDMs from mice stimulated with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P(Il1b, Ctr vs. Ctr+LPS) = 0.0013, P(Il1b, Ctr+LPS vs. Grx1 KO+LPS) = 0.0091, ***P < 0.0001. j mRNA levels of Il1b, Il6, and Tnfα in BMDMs pre-treated with an FABP5 inhibitor (SBFI26, 100 μM) for 2 h and stimulated with LPS (100 ng/mL) for 4 h, as determined by qPCR, n = 3 in each group, ***P < 0.0001. k mRNA levels of Il1b, Il6, and Tnfα in BMDMs after treated with SBFI26 (100 μM) for 2 h and stimulation with LPS (100 ng/mL) for 4 h, as evaluated by qPCR, n = 3 in each group, P(Il1b, Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P(Il16, Ctr+LPS vs. Ctr+LPS+inhibitor) < 0.0001, P(Tnfα, Ctr+LPS vs. Ctr+LPS+inhibitor) = 0.0046. 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 (e, i-k). *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance. Source data are provided as a Source Data file.

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