Figure 2. Acid (pH 6.0) induces RIP1 phosphorylation and physical association between RIP1 and ASIC1a.
(A) Acid-induced phosphorylation of RIP1 and its inhibition by 40 μM Nec-1, as measured by 32P incorporation. (B) Acid-induced phosphorylation of RIP1 and its inhibition by Nec-1 (40 μM) and PcTX1 (50 nM), detected using the anti-phospho S/T antibody. (C) Acid failed to induce RIP1 phosphorylation in neurons from Asic1a−/− mice. Five-fold more proteins were loaded for Asic1a−/− samples than for Asic1a+/+ samples. (D1, D2) Time courses (D1) and representative ratio images (D2) of intracellular acidification of cultured mouse cortical neurons from Asic1a+/+ and Asic1a−/− mice in response to extracellular pH decrease from 7.4 to 6.0, monitored using BCECF (n=30 for each genotype, peak changes summarized in D1). (E) In vitro RIP1 phosphorylation assay in pH 7.4 and pH 6.0 reaction solutions. Bands for phosphorylated RIP1 are indicated by the arrowheads. (F1, F2) Acid altered RIP1 expression levels with time. Shown are representative blots (F1) and summary data (F2) (n=5, **p<0.01 vs pH 7.4, by paired t test). (G1, G2) pH 6.0 treatment caused association of RIP1 with ASIC1a (G1, IP (immunoprecipitation) RIP1, IB (immunoblotting) ASIC1a; G2, IP ASIC1a, IB RIP1). (H) PcTX1 disrupted acid-induced ASIC1a–RIP1 association. (I) TNF-α neutralizing antibody (1 μg/μl and 2 μg/μl) failed to rescue pH 6.0 solution-induced neuronal death (CTB assay, n=3, ***p<0.001 vs vehicle (Veh) at pH 7.4; n.s., no statistical significance, vs Veh at pH 6.0).
Figure 2—figure supplement 1. Controls for Figure 2G,H,I.
