J

J. as a nucleation site for the formation of WS3 signaling complexes, (ii) endosomal EGFR signaling is sufficient to activate the major signaling pathways leading to cell proliferation and survival, and (iii) endosomal EGFR signaling is sufficient to suppress apoptosis induced by serum withdrawal. The growth-stimulatory signal of epidermal growth factor (EGF) is definitely mediated from the transmembrane EGF receptor (EGFR). The binding of EGF in the cell surface induces dimerization of EGFR, which results in the activation of EGFR tyrosine kinase activity and for WS3 5 min to remove cell debris and nuclei (p1). The postnuclear supernatant (S1) was then centrifuged at 1,500 for 10 min to yield a supernatant (S2) and a pellet (P2). Next, P2 was resuspended in homogenization buffer (0.25 M sucrose), overlaid upon an equal volume of 1.42 M sucrose buffer, and centrifuged at 82,000 for 1 h. The pellicule in the 0.25-to-1.42 M interface was collected PRDI-BF1 as the PM fraction. The S2 portion was centrifuged at 100,000 for 30 min to yield the soluble CY portion and a microsomal pellet. This pellet was resuspended in 0.25 M sucrose buffer and overlaid on a discontinuous sucrose gradient containing equal volumes of homogenization buffer at 1.00 and 1.15 M sucrose. The resuspension was centrifuged at 200,000 for 1.5 h to obtain the purified EN fraction in the 0.25-to-1.00 M interface. For a typical WS3 experiment, the total yield is definitely 30 g for the plasma membrane, 30 g for the EN portion, and 1 mg for the CY portion. The total yields of each portion were very consistent for all the treatments. Indirect immunofluorescence. Indirect immunofluorescence was performed as explained previously (41). Briefly, cells were cultivated on glass coverslips and serum starved for 24 h. After treatment, the cells were fixed by methanol and permeabilized with 0.2% Triton X-100. Next, the cells were incubated with primary antibodies at space temp for 1 h followed by fluorescence-labeled secondary antibodies for 1 h. Immunoprecipitation and immunoblotting. Immunoprecipitation experiments were carried out as explained previously (41). Cells were lysed with immunoprecipitation buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, 100 mM NaF, 0.5 mM Na3VO4, 0.02% NaN3, 0.1 mM 4-[2-aminoethyl]-benzenesulfonyl fluoride, 10 g of aprotinin/ml, 1 M pepstatin A) overnight at 4C. Cell lysates were then centrifuged at 21,000 for 30 min to remove debris. The supernatants, comprising 1 mg of total protein, were incubated with 1 g of mouse anti-EGFR antibody to immunoprecipitate EGFR from BT20 and MDCK cells. For control experiments, primary antibodies were replaced with normal mouse or sheep IgG WS3 (Sigma), and no EGFR was precipitated by normal IgG. Ras activation assay. Ras activation was assayed by the method explained by Herrmann et al. (20). Briefly, BT20 cells which had been treated as required were lysed and scraped into 0.5 ml of BOS buffer (50 mM Tris-HCl [pH 7.4], 200 mM NaCl, 1% NP-40, 10% glycerol, 10 mM NaF, 2.5 mM MgCl2, 1 mM EDTA) and then centrifuged at 21,000 and 4C for 30 min. Glutathione for: hr / /th th colspan=”6″ rowspan=”1″ align=”center” valign=”bottom” Control cells hr / /th th colspan=”6″ rowspan=”1″ align=”center” valign=”bottom” Cells treated with AG1478 + EGF + monensin hr / /th th colspan=”3″ rowspan=”1″ align=”center” valign=”bottom” Without EGF hr / /th th colspan=”3″ rowspan=”1″ align=”center” valign=”bottom” With EGF hr / /th th colspan=”3″.