Surface biotin was then stripped from your cells with a 10-min incubation in 50 mm MesNa in TBS (pH 8

Surface biotin was then stripped from your cells with a 10-min incubation in 50 mm MesNa in TBS (pH 8.6) twice, followed by washing and quenching of the MesNa BMS-5 with 20 mm iodoacetamide in TBS for 10 min. were transferred to a PVDF membrane (Millipore) and detected with the indicated main and secondary antibodies using an Immobilon Western Chemiluminescent HRP Substrate (Millipore), according BMS-5 to the manufacturer’s instructions. For immunoprecipitates, cells were lysed with detergent-free TBS buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl) by being passed through a 21-gauge needle as described previously (18). Briefly, cells were resuspended in the TBS with protease and phosphatase inhibitors and lysed by being exceeded through a 21-gauge needle 30 occasions. After centrifugation at 1,000 for 10 min, the supernatant was collected. The remaining pellet was again syringed 30 occasions. After centrifugation at 1,000 for 10 min, the second postnuclear supernatant was combined with the first, and protein concentrations were determined using a BCA protein assay BMS-5 kit. Comparative amounts (600 g) of the supernatants were immunoprecipitated with anti-GFP-agarose, anti-EGFR antibody, or cholera toxin B PRKMK6 subunit-biotin and Streptavidin-conjugated agarose for 1 h at 4 C with rotation, and then the immunoprecipitates were washed twice with lysis buffer and subjected to 6% SDS-PAGE. Cell Growth and Colony Formation Analysis The growth of the indicated cells was BMS-5 estimated by determination of cell growth curves or colony formation assays. To assay the cell growth curves, the cells (3 104) were seeded in 6-cm dishes overnight and then serum-starved for either 24 or 48 h (for MDA-MB-231 cells). After starvation, the cells were supplied with DMEM made up of 10% FBS with or without EGF (0.1 ng/ml), control IgG (10 g/ml), or anti-EGFR-blocking Ab (10 g/ml). The photographs of the same areas around the cultured dishes were taken at the indicated occasions (0, 24, 48, and 72 h), and the cell figures were counted. Cell figures were normalized to those at 0 h and statistically analyzed. To assay the colony formation, the control, GFP, WT, and S3C5 group cells (0.6 103) were seeded in the 6-cm dishes. Cells were incubated for 14 days to allow colony formation, cells were stained with 0.25% crystal violet for 15 min, and images were taken. Quantification of the colonies was obtained by measuring the for 10 min, the postnuclear supernatant (1 ml) was mixed with the same volume of the base buffer made up of 50% (v/v) OptiPrep and then added to the bottom of a 5-ml ultracentrifuge tube. Subsequently, each 0.6 ml of 20, 15, 10, 5, and 0% of OptiPrep in the base buffer was sequentially overlaid to the ultracentrifuge tube. The gradient was centrifuged at 5.2 104 rpm for 18 h at 4 C, using an ultracentrifuge (Hitachi himac CS100GX). A total of 12 fractions (0.4 ml for each) were carefully collected from top to bottom of the gradient and analyzed by WB, as explained above. Cell-spreading Assay The cell-spreading assay was performed as explained previously with minor modifications (33). Briefly, 6-well plates were coated with FN (10 g/ml) in PBS overnight at 4 C and then blocked with 1% bovine serum albumin (BSA) in DMEM for 1 h at 37 C. The indicated CHO-B2 cells were detached and suspended in serum-free DMEM with 0.1% BSA at 3 104 cells/ml. After replating around the FN-coated dishes for 20 min, non-adherent cells were removed by washing with PBS, and the attached cells were fixed with 4% paraformaldehyde in PBS, and representative photographs were then taken by phase-contrast microscopy. Immunofluorescence To assay the EGF-Alexa 555-based EGFR endocytosis, the indicated CHO-B2 cells were produced on coverslips (MatTek Corp., Ashland, MA) and starved for 24 h, followed by stimulation with a serum-free medium made up of 50 ng/ml Alexa Fluor? 555-conjugated EGF (EGF-555) and 0.3 mm BMS-5 primaquine, a recycling inhibitor, for the indicated occasions (0, 2.5, 5, 10, and 15 min). Cells were washed and fixed in 4% paraformaldehyde for 20 min at room temperature, followed by two.