Fig. 3
From: Malate initiates a proton-sensing pathway essential for pH regulation of inflammation
BiP and IRF2BP2 show direct binding affinity which is impaired by L-malate. (a) Strategy for identifying L-malate-responding protein-protein interactions with BiP. (b) Overall and notable readouts in the protein-protein interaction microarrays. (c) Scatterplot of signals from the protein microarrays of the BiP, BiP + L-malate (1 mM, pH adjusted to 7.2) and control groups. Proteins whose interaction with BiP were detected to be inhibited by L-malate are shown as blue dots, while proteins whose interaction with BiP were detected to be promoted by L-malate are shown as red dots. Size of circle indicates the absolute value of the logarithm of the ratios of the binding signal in the BiP+MA-treated group to the binding signal in the BiP-treated group to base two. In addition, protein IRF2BP2 was labeled. (d) Top predicted upstream regulators of DEGs analyzed by RNA-seq in BMDMs treated with LPS compared to BMDMs treated with LPS plus L-malate (24 h). (e) Canonical pathway analysis of the DEGs from RNA-seq in BMDMs treated with LPS compared to that treated with LPS plus L-malate (12 h), which of interest were shown. (f) BIAcore diagram of IRF2BP2 (concentrations indicated with colored lines) and BiP (chip-coupled) with or without L-malate (6 mM, pH 7.4). (g) BIAcore diagram of IRF2BP2 (chip-coupled) and L-malate (pH 7.4, concentrations indicated with colored lines). (h) The red spots represent BiP-IRF2BP2 bindings detected by PLA assay in BMDMs treated with or without L-malate (6 mM, pH 6.7), under stimulation of LPS for 3 h. Nuclei are stained with DAPI. Scale bars, 20 μm. Representative data from three experiments. (i) Immunofluorescent staining showing BiP-IRF2BP2 co-localizations in LPS-stimulated BMDMs treated with or without L-malate (6 mM, pH 6.7) for 1 h. Nuclei are stained with DAPI. Scale bars, 10 μm. Representative data from three experiments. (j and k) Cellular BiP-IRF2BP2 interactions shown by Western blot analysis of co-immunoprecipitation in MG132-pretreated (5 μM, 0.5 h) Raw264.7 cell lines treated with MG132 (0.5 μM) plus L-malate (6 mM) for 3 h in the medium pH 6.7. (l) pHi of MG132-pretreated (5 μM, 0.5 h) Raw264.7 cell lines treated with MG132 (0.5 μM) plus L-malate (6 mM) for 1-2 h in the medium pH 6.7. n ≥ 3. The data are shown as the mean ± SEM. *p < 0.05; **p < 0.01, ***p < 0.001. (two-sided Mann‒Whitney U test, unpaired Student’s t-test, Mantel‒Cox survival analysis). See also Supplementary Fig. 9
