Characterization of TNFR1 shedding from ARTS-1 cell lines. (a) ARTS-1 expression does not alter TACE protein levels. Immunoblots were performed on membrane fractions of ARTS-1 cell lines, as described in the legend to Figure 5a, and reacted with an anti-TACE antibody. Samples 1 and 2 are from representative clone cell lines. (b) ARTS-1 expression does not alter TNFR1 mRNA levels. Ribonuclease protection assays were performed on total RNA isolated from ARTS-1 cell lines. Probe, undigested riboprobe; Y, yeast tRNA negative control. (c) ARTS-1 expression does not alter TNFR1 subcellular localization. Crude membrane fractions from ARTS-1 cell lines, with or without TAPI-2 (25 μm) treatment, were centrifuged through a discontinuous sucrose gradient. TCA-precipitated proteins were immunoblotted with antibodies against TNFR1, β-catenin, and GM130. Discontinuous sucrose gradient fractions are as follows: Lane 1, 0.25 M; lane 2, 0.25/0.5 M interface; lane 3, 0.5 M; lane 4, 0.5/0.86 M interface; lane 5, 0.86 M; lane 6, 0.86/1.15 M interface; lane 7, 1.15 M; lane 8, 1.15/1.4 M interface; lane 9, pellet. (d) Increased TNFR1 shedding is preserved in ARTS-1 catalytic site mutants. Cell culture supernatants were collected after 24 hours and the amount of sTNFR1 present was determined by ELISA (n = 5). *P < 0.02 compared with ARTS-1. (e) TAPI inhibits ARTS-1–mediated increases in TNFR1 shedding. ARTS-1 cell lines were treated for 24 hours with TAPI-1 or TAPI-2 (25 μM). The amount of sTNFR1 present in cell culture supernatants was determined by ELISA and compared with untreated cells (n = 5). * P < 0.05.