Mutation of the tyrosine based sorting motif abrogates surface expression of the 3a protein. (A) Distribution of 3a protein in different cell lines - COS-7, HT29, MDCK and Huh7. The pSGI-3A-HA construct was transfected into these cell lines and stained with rabbit anti-3a antibody and Alexa-594 conjugated anti-rabbit IgG. (B) Surface expression of 3a and its mutants - Huh7 cells were transfected with expression constructs for wild type or mutant 3a proteins. Protein expression on the cell surface was detected using an anti-Myc antibody without permeabilizing the cells; the total levels were detected with anti-3a antibody after permeabilizing the cells with methanol. Arrow indicates surface distribution. Data shown are representative of three different experiments.