Affinity-purified anti-Syt IV antibody does not react with other Syt isoforms. (A) Five hundred nanograms of each soluble Syt I–VIII-GST fusion protein were subjected to SDS-PAGE and analyzed by Western blotting. Filters were probed with the affinity-purified Syt IV antibody in the absence (A, top panel) or presence (A, middle panel) of 500 μg of soluble Syt IV. The presence of GST-Syt fusion proteins was confirmed by probing a filter with an anti-GST mAb (A, bottom panel). (B) Cell lysates from COS-7 cells transiently transfected with full-length Syt IV cDNA cloned into the eukaryotic expression vector pcDNA3.1 zeo+, in the forward (+) and reverse (−) orientations, were examined for expression of Syt IV by Western analysis. Filters were probed in the absence (B, top panel) or presence (B, bottom panel) of 500 μg of soluble Syt IV. (C) PC12 cells treated as indicated were examined for Syt IV expression by Western analysis. Filters were probed with anti-Syt IV antibody in the absence (C, top panel) or presence (C, bottom panel) of 500 μg of soluble Syt IV.