J. (C4-CER A1, BPR1J-097 A2, or A3). For blockade of CER metabolic pathways, CFPAC-1 cells were pretreated for 4 h with 10 m d-MAPP (Biomol) (26), 2 m DMS (Enzo Life Sciences) (27), 20 m fumonisin B1 (Sigma-Aldrich) (28), 10 m d-PDMP (Biomol) (29), or 1 m NVP-231 (Sigma-Aldrich) (30), followed by a 20-h incubation with vehicle or 10 m C4-CER in the presence or absence of the above CER metabolic pathway inhibitors. For inhibition of PI3K kinase activity, CFPAC-1 cells were pretreated for 4 h with 0.2 m wortmannin (Calbiochem) (31), 8 m LY294002 (Calbiochem) (32), or 2 m PI-103 (Tocris Bioscience) (33), followed by a 20-h incubation with vehicle or 10 m C4-CER in the presence or absence of the above PI3K inhibitors. To block PDK1 or SGK1 kinase activity, the cells were pretreated for 4 h with various concentrations of PDK1 inhibitor GSK2334470 BPR1J-097 (Tocris Bioscience) (34) or SGK1 inhibitor GSK650394 (Tocris Bioscience) (35), followed by a 20-h incubation with vehicle or 10 m C4-CER in the presence or absence of various concentrations of GSK2334470 or GSK650394, respectively. All chemical inhibitors were prepared in ETOH or DMSO. After treatments, cells were subjected to cell lysis or cell surface biotinylation, followed by Western blot analysis. Cycloheximide Chase CFPAC-1 or CFBE cells were BPR1J-097 pretreated at 37 C for 20 h with C4-CER or were grown at 27 C for 48 h, to induce maturation of F508-CFTR. Pretreated cells were then incubated at 37 C with 20 g/ml cycloheximide (CHX; Sigma-Aldrich) for the indicated times (0, 2, 4, and 8 h). At the end of each indicated time period, cells were subjected to cell lysis or cell surface biotinylation, followed by Western blot analysis. siRNA Transfection The following SMART-pool siRNAs were obtained from Ambion: PI3K catalytic subunits (p110) and , PDK1, SGK1, Rictor, Lamin A/C, and scrambled control. Transfection of all siRNAs (50 nm) to CFPAC-1 cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfected cells were then grown at 37 C for 48 h in culture medium, followed by a 20-h treatment with vehicle or 10 m C4-CER. The cells were then subjected to cell lysis or cell surface biotinylation, followed by Western blot analysis. Cell Surface Biotinylation Assay Chemically treated or siRNA-transfected cells were rapidly washed with cold PBS (pH 8.2) solution supplemented with 1 mm CaCl2 and 1 mm MgCl2 and then subjected to cell surface biotinylation using sulfo-NHS-SS-biotin (Pierce) as described previously (10). After cell lysis, biotinylated proteins from the cell lysates were pulled down using streptavidin-agarose (Pierce) and were eluted in Rabbit polyclonal to Wee1 Laemmli SDS-PAGE sample buffer supplemented with 50 mm dithiothreitol, followed by SDS-PAGE and Western blot analysis. Cell Lysis and Western Blotting Cells were lysed in radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1% (v/v) Nonidet P-40, 1% (v/v) SDS) supplemented with HaltTM protease/phosphatase inhibitors (Pierce), followed by sonication and centrifugation at 14,000 for 10 min at 4 C. Protein concentrations were determined according to the BCA method (Pierce). Equal amounts of protein from the cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in Tris-phosphate saline buffer containing 5% (w/v) dry milk and 0.5% (v/v) Tween 20. After incubation with the primary antibody overnight at 4 C, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody BPR1J-097 (Jackson ImmunoResearch). Immunoreactive proteins were detected using Immobilon enhanced chemiluminescence (ECL; Millipore), and the signals were captured by a FujiFilm LAS-1000 system and quantitated by FujiFilm Image Gauge version 3.0. The quantitated values of CFTR band B and band C or surface CFTR were normalized to the corresponding values of -actin detected in the same cell lysates. CFTR Immunoprecipitation Equal amounts of total protein from the cell lysates were precleared with protein A-immobilized Dynal beads BPR1J-097 (Invitrogen). The resulting lysates were then incubated with mouse anti-CFTR C-terminal antibody for 4 h, followed by overnight incubation at 4 C with protein A-immobilized Dynal beads. IgG from the same species as the antibody being used for the immunoprecipitation was used as a negative control. Immunoprecipitated proteins were eluted in Laemmli SDS-PAGE sample buffer and separated by SDS-PAGE, followed by Western blot analysis. In Vitro Phosphorylation of CFTR Wild-type and F508-CFTR immunoprecipitates from untreated repaired and CFPAC-1 cell lysates, respectively, were.