Characterization of antibodies against S3 and S4. (A) Organization in three sub-regions, C1, C2 and V (upper panel), and amino acid alignment of the cytoplasmic C-terminal regions of human S1–S4. Antibodies were raised against the entire cytoplasmic tails of S3 or S4, and purified by affinity on peptides encompassing only the variable (V) regions (underlined peptide sequences). Dashes represent gaps in the sequence introduced to optimize alignment. The position of the residues in the entire sequence of the proteins is indicated (aa). TM, transmembrane region. (B-E) Specificity of S3 and S4 antibodies was tested by immunoblotting (B, D) and immunocytochemistry (C, E) using COS-7 cells transfected with GFP-fusion proteins expressing either the intracellular regions of S1 to S4 (B-E, S1–S4) or only the variable regions of S3 and S4 (B and D, S3V and S4V). Antibodies directed against GFP (anti-GFP) were used to verify the expression of the proteins. COS-7 cells transfected with the vector alone were used as mock control in immunoblotting experiments (-). Anti-S3 and anti-S4 antibodies recognized specifically GFP-fusion proteins encompassing S3 and S4 intracellular region, respectively. Scale bars: 20 μm.