Ecl luminata forte

Protein was extracted from the kidney and mouse mesangial cells (MCs) by RIPA buffer (Boston BioProducts, Worcester, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA). After sonication, the protein lysate was centrifuged at 12,000× g at 4 °C for 10 min. Protein concentrations of the samples were determined by Bradford assay. An equal amount of protein extract (25 μg) was electrophoresed by SDS-PAGE and immunoblotted overnight onto the polyvinylidine difluoride (PVDF) membrane. The membranes were blocked with 5% non-fat dry milk in TBST for 1 h at room temperature, washed three times in TBST, and subsequently incubated overnight with the respective primary antibodies at 4 °C. Membranes were washed three times in TBST to remove the unbound primary antibody and then incubated with corresponding HRP-conjugated secondary antibodies for 2 h at room temperature. Thereafter, the membranes were washed and the immunoreactive protein bands were developed using ECL LuminataForte (Millipore, Temecula, CA, USA), visualized under a ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) and quantified by densitometric analysis using ‘ImageJ’ software.

Cell lines were seeded into six-well plates (10⁶ cells per well) with increasing concentrations for the indicated drugs. Twenty-four hours later, cells were stimulated with 100 ng/ml neuregulin (NRG, Cell Signaling, Danvers, MA) for 10 min at 37 °C. Subsequently, cells were lysed using RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% Na-deoxycholate, 1% NP-40, 2 mM EDTA) followed by sonification. Finally, 20 μg of whole-cell protein lysate was resolved on a 10% Bis-Tris gel (Roth, Karlsruhe, Germany) at 30 mA per gel. Proteins were transferred by wet blotting (Criterion™ blotter, Bio-Rad, Hercules, CA) onto a PVDF membrane (Merck-Millipore, Billerica, MA), and probed with primary antibodies p-Akt (1:1000; Cell Signaling), Akt (1:1000; Cell Signaling), and Actin (1:2000; Sigma, Saint Louis, MO) at 4 °C over night. On the next day, membranes were incubated for 2 h with the secondary antibody goat-anti-rabbit-HRP (1:30,000; Acris, Hiddenhausen, Germany). Signals were detected using ECL Luminata forte (Merck-Millipore) and a CCD camera system (LAS 4000mini; GE Healthcare, Munich, Germany).

Samples were lysed in BEX Lysis buffer (25 mM Tris pH 8.0, 20 mM NaCl, 0.6% w/v Deoxycholate, 0.6% Igepal CA-630) containing 1x HALT protease and phosphatase inhibitor cocktail (Pierce) and centrifuged for 15 min at 4°C and >13,000 g. Lysates were electrophoresed under denaturing conditions using NuPage Novex Bis-Tris 4-12% gradient gels and MES running buffer (both Invitrogen) according to the manufacturer's instructions. Proteins were transferred onto PVDF membranes (Roche) by wet blot using XCell II Blot Modules (Invitrogen). Membranes were blocked in 1x RotiBlock (Roth) for 1 h. Incubation with primary antibodies was performed overnight at 4°C in 1x RotiBlock. On the next day, after three 10 min washing steps in PBS-T, blots were incubated with HRP-coupled secondary antibodies for 1 h at room temperature. After three washing steps in PBS-T, blots were analyzed using the LAS-4000 CCD imager (GE Healthcare) and Luminata forte ECL (Millipore). Densitometric analysis was performed using ImageQuantTL (GE Healthcare).