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The purity of the samples was at least 80%, as judged by Coomassie Brilliant Blue staining of SDS-polyacrylamide gel electrophoresis (PAGE). 2.5. GST Pull-Down Assay and Western Blotting Cells were washed with ice-cold phosphate-buffer saline (PBS) and suspended in lysis buffer containing 25?mM Tris-HCl (pH 7.2), 150?mM NaCl, 5?mM MgCl2, 1% NP-40, 5% glycerol, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The cell lysates were briefly sonicated at 4��C and separated from the pellets after centrifugation at 12,100?g for 15 minutes. Protein concentrations were estimated with the BCA protein assay reagent. Glutathione-sepharose beads coupled with GST, GST-ANKR, or GST-GGA3 were incubated for 2 hours at 4��C with 500?��g of the cell lysates. After washing the beads four times with lysis buffer, proteins bound to the beads were analyzed by 10% or 12.5% SDS-PAGE followed by western blotting. The samples were subjected to SDS-PAGE and transferred to the PVDF membrane (Bio-Rad, Richmond, CA). Western blotting was carried out using a polyvinylidene difluoride (PVDF) membrane and the ECL Prime Western Blotting Detection System (GE Healthcare, Piscataway, NJ). The membrane was blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 30 minutes at room temperature and probed with primary antibody at 4��C overnight. After washing, the membrane was incubated with horseradish conjugated with anti-rabbit or anti-mouse secondary antibody (dilution 1?:?10,000) for 2 hours at room temperature, developed using an ECL Prime reagent and exposed to Hyperfilm (GE Healthcare, Piscataway, NJ). The following primary antibodies were used: anti-ARF6 antibody (1?:?1,000) and anti-GFP antibody (1?:?2,000). The intensity of the protein bands was measured using Photoshop CS5 software. 2.6. Phagocytosis Assay Zymosan was suspended in PBS and boiled for 30 minutes. After a brief washing, zymosan was resuspended in PBS (10?mg/mL) and sonicated for several minutes to disperse the particles prior to use. For the quantitative assay of phagocytosis, zymosan was added to adherent RAW264 macrophages. After 30 minutes of Sitaxentan incubation with zymosan at 37��C, the cells on the coverslips were fixed with 4% paraformaldehyde. The number of internalized zymosan particles was counted in 50 cells randomly chosen under a phase-contrast and fluorescence microscope. The phagocytic index, that is, the mean number of zymosan particles taken up per cell, was calculated. The index obtained for the transfected cells was divided by the index obtained for the untransfected (control) cells and expressed as a percentage of the control cells. 2.7. Live-Cell Imaging and Image Analysis RAW264 cells were cultured onto 25?mm circular coverslips and assembled in an RB-filled chamber on the thermocontrolled stage (Tokai Hit, Shizuoka, Japan).