Fig. 8. Endogenous NRADD is a RIP substrate. (A) NRADD processing in vivo. NIH3T3 cells were scraped from the tissue culture dish and incubated at 37°C for 3 hours in the presence of the indicated inhibitors. The first lane (4°C) shows cells kept at 4°C after scraping and lysed immediately. Cell lysates were analysed by anti-NRADD western blot. -Secretase substrate (NpNR) and product (-PNR) are indicated. (B) NRADD processing in vitro. N2a cell membranes were isolated and incubated at 37°C for 1 hour with the indicated inhibitors. (C) Subcellular fractionation. N2a cells were treated for 40 minutes with APMA or left untreated, then washed and incubated for 3 hours in the presence of lactacystin (10 µM). Cells were harvested and fractionated as described in Materials and Methods. N, Nuclear fraction; S100SN, Cytosol; X100P, Triton-X-100-insoluble fraction; X100SN, 1% Triton-X-100-soluble membranes (n=3). (D) Detection of ECD in conditioned medium. 293 cells were transfected with NRADD tagged at the N-terminus with AU1 (AU1 NR) or vector (Vect.). 18 hours after transfection PMA (100 ng ml^-1) was added as indicated and, after 70 hours, medium was harvested and cell lysates prepared. Lysates and conditioned medium were PNG-F treated. Conditioned media were immunoprecipitated with anti-AU1 antibody and analysed by anti-AU1 western blot of a 16% Tricine gel. ^*NRADD truncated at the C-terminus.