Characterization of ARTS-1 as a type II integral membrane protein. (a) Specificity of anti–ARTS-1 serum. Immunoblots were performed on membrane and cytosolic fractions from NCI-H292 cells using anti–ARTS-1 immune or preimmune serum. Competitive inhibition experiments were conducted by preincubation of anti–ARTS-1 immune serum with either BSA or the peptide epitope against which the anti–ARTS-1 immune serum was raised. (b) ARTS-1 is a membrane-associated protein. Membrane (M) and cytosolic (CY) protein fractions of human bronchial epithelial cells (HBECs) obtained via bronchial brushings (left panel), human bronchial epithelial cell lines (NCI-H292, BEAS-2B, BET-1A, and A549) (center panel), and primary cultures of normal human bronchial epithelial cells (NHBEs), HUVECs, and fibroblasts (right panel) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti–ARTS-1 immune serum. (c–f) Colocalization of membrane-associated ARTS-1 and TNFR1 in human bronchial epithelial cells. Confocal immunofluorescence laser microscopy was performed on nonfixed, nonpermeabilized frozen sections of normal human bronchi (c and d) and on nonfixed, nonpermeabilized cytospin preparations of normal human bronchial epithelial cells obtained via bronchial brushings (e and f) using a murine IgG_2b isotype control and preimmune serum (c and e) and anti-TNFR1 and anti–ARTS-1 antibodies (d and f). An annotated differential interference contrast image is shown in the bottom left panels. Arrows denote the apical cell membrane. C, cilia; BM, basement membrane; SM, submucosa; N, nucleus; L, lateral cell membrane; B, basal cell membrane.