Antibodies were raised in rabbits against two peptides within the luminal domain of BHKp23 (antibodies against luminal peptide 1 [LP1]: KNYEEIAKVE, and against luminal peptide 2 [LP2]: RLEDLSESIVNDFAY), and against a peptide corresponding to the cytoplasmic tail of BHKp23 (CT: TWQVFYLRRFFKAKKLIE). For affinity purification, the antigens were coupled to Affi Gel 10 (Bio-Rad Laboratories, Hercules, CA) as described by the manufacturer; antibodies were then bound to the coupled antigen in 10 mM Tris, pH 7.4, and eluted with 0.1 M glycine, pH 2.5. Antibodies were biotinylated with biotinyl-ε-aminocaproic acid N-hydroxysuccinimide ester (Gruenberg and Gorvel, 1993 ). Fab fragments of antibodies were prepared with papain immobilized on beads (Pierce Chemical Co., Rockford, IL) according to the instructions of the manufacturer. Rabbit antisera against the EAGE epitope of β-COP (Pepperkok et al., 1993 (link)), the mammalian KDEL receptor, ERD2 (Griffiths et al., 1994 (link)), and TGN38 (Luzio et al., 1990 (link)) were gifts of T.E. Kreis (University of Geneva, Geneva, Switzerland), H.D. Söling (University of Göttingen, Göttingen, Germany), and G. Banting (University of Bristol, Bristol, United Kingdom), respectively. Mouse mAb against β-COP (maD: Pepperkok et al., 1993 (link); and M3A5: Allan and Kreis, 1986 (link)), ERGIC-53 (G1/93: Schweizer et al., 1988 (link)), and β′-COP (CM1A10: Orci et al., 1993 (link); Lowe and Kreis, 1996 (link)) were provided by T.E. Kreis, H.P. Hauri (University of Basel, Basel, Switzerland), and J. Rothman (Sloan-Kettering Institute, New York), respectively. Mouse mAbs that recognize the myc epitope (9E10: Evan et al., 1985 (link)), a cytoplasmic epitope of VSV-G (P5D4: Kreis, 1986 (link)), and an exoplasmic epitope of VSV-G (17.2.21.4: Gruenberg and Howell, 1985 (link); and VG: Pepperkok et al., 1993 (link)) have been described. Fluorescein-labeled, anti–mouse IgG, rhodamine-labeled, anti–rabbit IgG, and fluorescein- labeled streptavidin were from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Unspecific control rabbit IgGs were from Sigma Chemical Co.