Fig. 6
From: Malate initiates a proton-sensing pathway essential for pH regulation of inflammation
BiP-IRF2BP2 signaling senses intracellular protons independently of lysosomal pH. (a and b) Cellular BiP-IRF2BP2 interactions shown by Western blot analysis of co-immunoprecipitation in MG132-pretreated (5 μM, 2 h) peritoneal macrophages which were incubated with Nigericin (10 μM), Valinomycin (10 μM) and MG132 (5 μM) for 3 h in calibration buffer under different pH (7.2, 7.0, 6.8, 6.6). (c and d) Cellular BiP-IRF2BP2 interactions shown by Western blot analysis of co-immunoprecipitation in MG132-pretreated (5 μM, 0.5 h) Raw264.7 cells which were treated with acidized medium (AcM) (pH 6.7) or with AcM plus monensin (0.1 μM) in the presence of LPS and MG132 (0.5 μM) for 6 h. (e and f) IRF2BP2 protein levels visualized (e) and quantified (f) by Western blot in CHX-treated (6 h) BMDMs with or without acidized medium (AcM) (pH 6.7) and monensin. (g and h) Cellular BiP-IRF2BP2 interactions shown by Western blot analysis of co-immunoprecipitation in MG132-pretreated (5 μM, 0.5 h) Raw264.7 cells which were incubated by Ringer’s solution containing glucose (11 mM), MG132 (0.5 μM) with or without NH4Cl (30 mM) for 2 h. (i and j) Cellular BiP-IRF2BP2 interactions shown by Western blot analysis of co-immunoprecipitation in MG132-pretreated (5 μM, 0.5 h) Raw264.7 cells which were treated MG132 (0.5 μM) by with or without ArA (200 μM, neutralized pH) for 6 h. (k) IRF2BP2 protein abundance was visualized by immunoblotting in BMDMs isolated from Hspa5f/f or Hspa5f/f; Lyz2-Cre mice. These CHX-treated BMDMs were treated with/without ArA or DB (9 h). (l and m) IRF2BP2 protein levels in BMDMs isolated from Hspa5f/f or Hspa5f/f; Lyz2-Cre mice. These LPS-stimulated BMDMs were treated with/without AcM (pH 6.7) or ArA (8 h). n ≥ 3. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (unpaired Student’s t-test)
