Fig. 5: NSF knockdown protects against acidotoxicity in cultured neurons.

a, b Knockdown efficiency of NSF shRNA in cultured neurons determined by western blotting. Shown are representative blots (a) and summary data (b). n = 3, *** p < 0.001 vs. Scrm (scrambled shRNA) by paired t test. c shRNA knockdown of NSF attenuated acid-induced associations of ASIC1a with NSF and ASIC1a with RIPK1. d Summary data of NSF pulled down by the ASIC1a antibody. The data are normalized to NSF/ASIC1a of neurons treated with Scrm at pH 7.4. n = 3, *** p < 0.05 vs. corresponding pH 7.4, ^## p < 0.01 vs. Scrm in pH 6.0, by ANOVA. e NSF knockdown attenuated acid-induced decrease of CFP-ASIC1a-YFP FRET. Similar to Fig. 2b, but the cells were transfected with Scrm or NSF shRNA as indicated. n = 11 cells for each. f Summary of ratio changes at 5 min of the pH 6.0 treatment for data in (e). *** p < 0.001 vs. CFP-ASIC1a-YFP with Scrm by unpaired t test. g PI staining of cultured cortical neurons prepared from WT and ASIC1a KO mice transfected with NSF-shRNA or Scrm shRNA. Scale bar, 50 µm. The neurons were treated at either pH 7.4 or pH 6.0 for 1 h and then returned to the normal culture medium for 24 h before PI staining. Knocking down NSF reduced acid-induced neuronal death in WT, but not ASIC1a KO, neurons. h Summary data for (g). n > 200 neurons counted for each. *** p < 0.001 vs. corresponding pH 7.4, ns, no statistical significance, ^### p < 0.001 vs. Scrm in pH 6.0, by ANOVA. i The nonspecific NSF inhibitor, NEM (3 μM), showed a protective effect on acid-induced neuronal death. n = 4, *** p < 0.001 vs. corresponding pH 7.4, ^### p < 0.001 vs. vehicle (DMSO) pH 6.0, by ANOVA. Error bars are SEM for all summary panels. The Scrm sequence used was CTTAAGGTTAAGTCACTCT.