Characterization of VAMP4 in AtT-20 cells.A, RT-PCR analysis performed on AtT-20 cDNAs with primers specific to VAMP4 (lane 2). Lane 1, Molecular weight standards; lane 3, negative control performed with no added DNA template; lanes 4, 5, controls performed with plasmids harboring either a VAMP2 cDNA or a VAMP4 cDNA as the template. A single band of 426 bp was observed only in reactions performed with either AtT-20 cDNAs or the VAMP4 plasmid. Digestion of this product with HindIII gave the predicted restriction pattern as deduced by the rat VAMP4 sequence. B, Immunoblot analysis of endogenous and transfected VAMP4. PNS was prepared from wild-type AtT-20 (wt, lane 1) and cells stably transfected with a FLAG-tagged VAMP4 plasmid (FV4, lanes 2, 3). Lysate (10 μg) was subject to SDS-PAGE and immunoblot analysis with either polyclonal antisera against a soluble form of recombinant rat VAMP4 (lanes 1, 2) or M2 monoclonal antibody against the FLAG epitope (lane 3). The anti-VAMP4 antisera recognized a single band of ∼18 kDa in wild-type cells and an additional band that also was recognized by the anti-FLAG antibody in FLAG–VAMP4 transfected cells. C, Colocalization of VAMP4 with ACTH immunoreactivities in the Golgi region, but not in the process tips. AtT-20 cells stably transfected with FLAG–VAMP4 were processed for indirect immunofluorescence. Fixed and permeabilized cells were doubly stained with anti-FLAG M2 monoclonal antibody (Cb, Ce) and anti-ACTH (Ca, Cd) polyclonal antibodies.Ca–Cc, A representative field of cells visualized by an Axiophot microscope, showing colocalization of staining patterns in the cell body (arrowheads), but not in the process tips (arrows) where MSGs accumulated. More than 50 cells were examined, and all show similar patterns. Cd–Cf, Higher resolution analysis of the staining patterns in the Golgi region by confocal microscopy. Scale bar, 10 μm.