Article Title: Analysis of the Membrane Topology of the Acid-sensing Ion Channel 2a
Figure Lengend Snippet: a, the effect of Endo H and PNGase on ASIC2a receptor protein. HEK293 cells transiently expressing the ASIC2a protein were prepared and treated with either Endo H or PNGase. The proteins were separated by SDS-PAGE, transferred to nylon membrane, and the resultant blot was probed with ASIC2a primary antibody (Alomone). The immunoblot shows that both Endo H and PNGase reduced the molecular mass of ASIC2a by ~5 kDa, implying that two glycosylation sites may exist for ASIC2a. b, ASIC2a contains two N-linked glycosylation sites. Mutations N365S and N392S produced ASIC2a channels with a reduced molecular mass, indicating they are both sites for N-linked glycosylation. A double mutant of N365S/N392S produced a reduced molecular mass equal in size to that of PNGase-treated ASIC2a wild type, indicating that only these two sites are required for full glycosylation of ASIC2a. c, the three remaining predicted N-linked glycosylation sites are not glycosylated. Immunoblot analysis of N22S, N478S, and N487S showed no reduction in molecular mass; thus, these ASIC2a proteins are not glycosylated in HEK293 cells. NT, non-transfected.
Article Snippet: The epitope of the ASIC2a antibody (Alomone) is directed against amino acids 1–20 of the intracellular amino terminus; thus, we compared ASIC2a expression in permeabilized versus non-permeabilized cells.
Techniques: Expressing, SDS Page, Western Blot, Produced, Mutagenesis, Transfection