Perk and terk

Cell lysate protein was quantified with a modified BCA protein assay using manufacturers protocol (Bio-Rad). Samples (20–30 μg) were ran on precast gels (10% Bis–Tris, Bolt brand, from ThermoFisher). Gels were wet-transferred to nitrocellulose membranes at 30 V for at least 60 min at 4 °C. Blots were blocked with 5% non-fat dry milk in TBS for 30 min, washed 3 × for 5 min with TBS + 0.1% Tween-20 (TBST), and incubated with primary antibody overnight at 4 °C. Primary antibody: pERK and tERK (Cell Signaling) at 1:1000 dilution in 5% BSA in TBST. Blots were then washed 3 × with TBST then incubated with secondary antibodies for 90 min at room temperature. Secondary antibodies: Goat anti-Rabbit 800 W and Goat anti-Mouse 680 diluted at 1:10,000 and 1:20,000 (Licor), respectively, in 5% non-fat milk in TBST. Blots were washed 3X with TBST and 1 × with TBS, then imaged on a Licor Odyssey Fc or Azure Sapphire. Bands were analyzed using Image-J and reported as pERK/tERK normalized to the standard, or simply as pERK/tERK.

AngII, losartan, enalapril and the antibody against Flag were purchased from Sigma-Aldrich (St. Louis, MO, USA). PD123319 was obtained from Selleckchem (Houston, TX, USA). Antibodies against COMP (Cat# ab42225, for western blot) and total p38-MAPK were obtained from Abcam (Cambridge, UK). The antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-arrestin-2, p-p38, phosphorylated and total ERK (p-ERK and t-ERK), p- and t-Smad2/3, p- and t-JNK, p- and t-pan-PKC and AUF-1 were obtained from Cell Signaling Technology (Boston, MA, USA). The antibody against AT1 (Cat# 25343-1-AP) was obtained from Proteintech Group, Inc. (Rosemont, IL, USA).