Cell lines were plated at 1×106 cells per 10 cm tissue culture plate and allowed to proliferate for 48 hours. Samples were prepared using 0.5 ml per plate of a lysis buffer containing 10 mM Tris-base, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 μM sodium orthovanadate, 1% Triton X-100, freshly added protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, and 34.4 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and the phosphatase inhibitor microcystin-LR (500 nM). Samples were allowed to incubate on ice for 30 min followed by centrifugation at 16,000g for 10 min at 4°C. Protein concentration of the lysates was measured using the Pierce BCA Protein Assay (Thermo Scientific, Rockford, IL).
Western immunoblotting, image acquisition and quantification of results were carried out as described previously [25 (link)]. Primary antibodies were obtained from the following sources: PP6-C, Millipore, Billerica, MA; SAPS1, 4E-BP1 and p-PKCα(Ser657), Santa Cruz Biotechnology, Inc., Santa Cruz, CA; SAPS2 and SAPS3, Bethyl Laboratories, Inc., Montgomery, TX; p-S6(Ser235/236) and p-NDRG1(Thr346), Cell Signaling Technology, Danvers, MA. Immunoblot detection was by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ).
Western immunoblotting, image acquisition and quantification of results were carried out as described previously [25 (link)]. Primary antibodies were obtained from the following sources: PP6-C, Millipore, Billerica, MA; SAPS1, 4E-BP1 and p-PKCα(Ser657), Santa Cruz Biotechnology, Inc., Santa Cruz, CA; SAPS2 and SAPS3, Bethyl Laboratories, Inc., Montgomery, TX; p-S6(Ser235/236) and p-NDRG1(Thr346), Cell Signaling Technology, Danvers, MA. Immunoblot detection was by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ).