Characterization of polyclonal BIN1 antibody BSH3. (A) Schematic illustration of the exon structure of BIN1 and possible alternate splicing. The protein structures of BIN1 isoforms 6-10 generated by alternate splicing of exons 13-17, which encode the CLAP and MBD are depicted. Note that exons 18-20 are invariable in all BIN1 isoforms. (B) Immunoblot analysis of C-terminally FLAG-tagged BIN1 isoforms 6-10 expressed in HEK293 cells [expression plasmids were generously provided by Dr. Zhou, Merck Research Laboratories]. Blots were probed with pAb BSH3 or anti-FLAG mAb. BSH3 was raised against residues encoded by exons 17-20. The results show that BSH3 is capable of reacting with isoform 10, which lacks exon 17 encoded residues. Thus, this antibody reacts with epitopes common to all BIN1 isoforms, encoded by exons 18-20. N = NH2 terminus; BAR = Bin-amphiphysin-Rvs domain; PI = phosphoinositide binding domain; CLAP = clathrin/Adaptor protein 2 binding domain; MBD = MYC-binding domain; SH3 = SRC homology 3 domain. (C) Immunofluorescence analysis of antibody specificity. HEK293 cells transiently transfected with FLAG-tagged human BIN1 isoform 7 were stained with a combination of antibodies against the FLAG epitope tag or the indicated BIN1 antibodies. Wide-field images acquired on a Nikon TE2000 microscope using a 100X objective reveal that each BIN1 antibody strongly stains transfected cells. (TIF 1481 kb)