To examine whether SLHMG cells translate transcripts for VIP and cholinergic receptors, cultured cells were trypsinized, centrifuged, and resuspended in 2X reducing sample buffer (BioRad, Hercules, CA). Samples were heated at 95°C for 10 minutes, separated by SDS-PAGE on 10% Tris/glycine precast gels (Invitrogen) and transferred to nitrocellulose. Membranes for VIP receptor blots were blocked with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) containing 0.01% Tween-20, followed by incubation with a mouse monoclonal antibody specific for VIP receptor 1 (VPAC1; GenWay Biotech, San Diego, CA) or VIP receptor 2 (VPAC2; Millipore Corp., Billerica, MA) and HRP-conjugated, Fc-specific goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO). Membranes for muscarinic acetylcholine (mACh) receptor blots were blocked with 5% nonfat dry milk in PBS with 0.01% Tween-20, followed by incubation with rabbit polyclonal anti-mAChR M2 or M3 antibody (GenWay Biotech) and HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich). Proteins were visualized with chemiluminescent detection reagents (West Pico; Pierce Biotechnology, Rockford, IL). Human heart extract (Imgenex, San Diego, CA) and A431 human skin epidermal cell lysate (Santa Cruz Biotechnology, Santa Cruz, CA) were processed for immunoblot analyses as positive controls for the mACh and VIP receptors, respectively.