Fig. 5
From: Malate initiates a proton-sensing pathway essential for pH regulation of inflammation
L-malate protects IRF2BP2 from BiP-driven protein degradation. (a and b) IRF2BP2 protein levels in CHX-treated (0 h, 3 h, and 6 h) BMDMs incubated with Tunicamycin (0.1 μM) or Thapsigargin (0.1 μM) added 16 h before CHX addition. (c and d) IRF2BP2 protein levels visualized (c) and quantified (d) by immunoblotting of BMDMs isolated from Hspa5f/f or Hspa5f/f; Lyz2-Cre mice. These BMDMs were treated by CHX (3 h) and incubated with/without Tunicamycin (0.1 μM) or Thapsigargin (0.1 μM) added 16 h before CHX addition. (e and f) IRF2BP2 abundance was visualized by immunoblotting (e) and quantification (f) of IRF2BP2 in BMDMs from Hspa5f/f or Hspa5f/f; Lyz2-Cre mice. These BMDMs under a controlled medium pH (6.7) were treated by CHX and incubated with or without L-malate (6 mM, 6 h). (g) pHi of BMDMs (controlled medium pH 6.7) treated with CHX (2 h) and incubated with or without L-malate (6 mM, 6 h). (h and i) IRF2BP2 abundance was visualized by immunoblotting (h) and quantification (i) of IRF2BP2 in BMDMs treated by LPS for 0, 8 h and 24 h. n = 3. (j) pHi of BMDMs treated with acidized medium (pH 6.7, 3 h), MG132 (5 μM, 0.5 h) or CHX (6 h). (k) Il1b mRNA levels in LPS-stimulated BMDMs treated with/without L-malate (6 mM) or MG132 (0.5 μM). In addition, MG132 (5 μM) was added in MG132-treated groups 0.5 h before LPS stimulation. (l) Il1b mRNA expressions in LPS-stimulated BMDMs treated with/without L-malate (6 mM, medium pH 6.7) and bafilomycin A (BafA) for 24 h. (m and n) IRF2BP2 protein levels in unstimulated and LPS-stimulated BMDMs treated with L-malate (6 mM, medium pH 6.7) and MG132 (0.5 μM) for 8 h. In addition, MG132 (5 μM) was added in MG132-treated groups 0.5 h before LPS stimulation. (o and p) IRF2BP2 ubiquitination shown by Western blot analysis of co-immunoprecipitation in MG132-pretreated (5 μM, 0.5 h) Raw264.7 cells treated with MG132 (0.5 μM) and L-malate (6 mM) in medium pH 6.7 for 3 h. (q and r) IRF2BP2 ubiquitination shown by Western blot analysis of co-immunoprecipitation in MG132-treated (5 μM, 0.5 h) Hspa5+/+ or Hspa5+/− Raw264.7 cell line. The “X” means unknown proteins detected in immunoblotting visualizing IRF2BP2. 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)
