Anti etar

Renal cortex was homogenized and extracted in a cold buffer containing 0.1 mol/l Tris (hydroxymethyl) aminomethane HCl. The tissue extracts were then partially purified by ethanol extraction. Proteins from GENs or mesangial cells were isolated from using RIPA buffer (Thermo Scientific, USA). 50 μg of protein samples were isolated in 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, USA) by electroblotting. The membranes were blocked in 5% non-fat dried milk for 60 min at room temperature. For the detection of RhoA, the membrane and cytoplasm proteins were extracted from mesangial cells using Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology), then membrane and cytosolic proteins were separated on SDS-PAGE. The membranes were probed with first primary antibody anti-ETBR (Abcam, USA), anti-ETAR (Abcam, USA), anti-p-p65 (Invitrogen, USA), anti-p65 (Invitrogen, USA), anti-CTGF (Abcam, USA), anti-RhoA (Abcam, USA), anti-collagen IV (Abcam, USA), anti-Fibronectin (Abcam, USA), anti-p21 (Abcam, USA) and anti-β-actin (Invitrogen, USA) and incubated at 4°C overnight. After washing with PBST, membrane was cultivated with secondary antibody for 60 min at room temperature. β-actin was used as internal control.

Whole cell lysates were prepared using a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a mixture of protease and phosphatase inhibitors. Total proteins were subjected to SDS-PAGE, and the antibodies (Abs) used for the study were as follows: anti-ET_AR and anti-β-actin (C-11), purchased from Abcam (UK) and Santa Cruz Biotechnology (CA, USA), respectively; anti-p-p44/42 MAPK (Thr202/Tyr204), anti-p44/42 MAPK, anti-pAkt (S473), anti-Akt, anti-cleaved-PARP (Asp214), and anti-cleaved-caspase 7 (Asp198), were from Cell Signaling Technology (MA, USA). Anti-E-cadherin and anti-N-cadherin were purchased from BD Biosciences (CA, USA). Western blotting filters were developed using the ECL-plus detection system or, otherwise, the SuperSignal West Femto kit (Thermo Scientific, IL, USA). Western blots for ET_AR were quantified by densitometric analysis using ImageJ software.