Validation of human ASIC1 KO cells. (A) Sequencing of genomic DNA from HEK293T WT cells or ASIC1 KO cells of human ASIC1 gene’s second exon. Positions highlighted in yellow show that all alleles of the KO line have been frame-shifted. (B) Western blot of WT HEK293T cells, ASIC1 KO cells, and KO cells overexpressing human ASIC1a (OE). (C) pH 5–evoked whole-cell current densities from HEK293T WT (black) and KO (gray) cells. Raw traces are inset. pH 5–evoked peak current density at −60 mV holding potential: WT, 20 ± 3 pA/pF, n = 20 cells; KO pH 5.0, 0.51 ± 0.05 pA/pF, n = 20 cells; P < 1e⁻⁵, Mann–Whitney U test. Circles represent individual cells, and error bars depict SEM.