PICK1 increases surface levels of ASIC1a. A, HEK293T cells were transfected with GFP-ASIC1a and cell lysates were subjected to Western blot analysis. Purified antibody detected a strong band in cells transfected with ASIC1a and this band was blocked by pre-absorbing with antigen ASIC1-CT (ASIC1a C-terminal 60 amino acids fusion protein). As a position control, GFP antibody detected a band at the same position, indicating this band is GFP-ASIC1a. B, ASIC1a alone or ASIC1a together with either wild-type or mutant PICK1 were transfected into HEK293T cells as indicated. Surface ASIC1a proteins were isolated using surface biotinylation assay. Total and surface samples were resolved by SDS-PAGE and immuno-blotted with the same ASIC1 antibody shown in A. Co-expression with PICK1 increased the band intensity of surface ASIC1a. C, Quantification data from multiple experiments in B. Surface ASIC1a ratios, expressed as the percentage of ASIC1a on the surface relative to total ASIC1a, were determined by fitting surface ASIC1a to a standard curve obtained by quantifying different amounts of total ASIC1a. Wild-type PICK1 significantly increased surface ASIC1a (** p < 0.01). Both the BAR domain mutant PICK1 2K-E and the PDZ domain mutant PICK1 KD-AA abolished PICK1-induced increase in surface ASIC1a. D, The effect of PICK1 on glycosylation of ASIC1a. Mouse ASIC1a was transfected alone or together with PICK1 into CHO cells and the lysates were treated with PNGase F or EndoH and analyzed by Western blot. PICK1 increased the species of faster migrating ASIC1a on the gel. PNGase F treatment reduced almost all slowly migrating ASIC1a to the faster migrating species. In contrast, there was still a small fraction of slowly migrating species left with EndoH treatment.