Fig. 4: NSF binds to the distal N-terminus of ASIC1a.
a Glutathione-S-transferase (GST) pull-down assay showing the pulldown of NSF by GST-NT, but not GST-CT. Purified GST, GST-ASIC1a C-terminus (GST-CT) or GST-ASIC1a N-terminus (GST-NT) were incubated with mouse brain extracts, immobilized on glutathione-Sepharose 4B beads, and immunoblotted for NSF (top) and GST (bottom). GST alone served as a negative control. b Amino acid sequences of NT deletion mutants of ASIC1a. c Co-IP experiments showing that deletion of aa1-41 (Δ1-41) or aa1-20 (Δ1-20) decreased the acid-induced association between ASIC1a and NSF in CHO cells. d Summary data of (c). n = 3, ** p < 0.01 vs. corresponding pH 7.4, ### p < 0.001 vs. WT, by ANOVA. e Co-IP experiments showing that NT1–20 (10 μM, 0.5 h pretreatment) reduced acid-induced formation of NSF-ASIC1a-RIPK1 complex in cultured mouse cortical neurons. NT1–20 also decreased acid-induced degradation of RIPK1 protein (lower panel). f Summary data of (e). n = 3, * p < 0.05, ** p < 0.01 vs. corresponding pH 7.4, # p < 0.05 vs. WT, by ANOVA. Error bars are SEM for all summary panels.
