Ecl plus western blotting reagent

Fifty µg of total protein isolated from RASMC-miR483 cells was used for the Western blotting assay. Fractions were prepared as above. The membrane was incubated overnight in primary antibody specific for AGT (Cat. No. ab108334; Abcam, MA, USA), ACE-1 (Cat. No. ab134709; Abcam), ACE-2 (Cat. No.AF933 R & D Systems, MN, USA and Cat. No. ab108252; Abcam) or AGTR2 (Cat. No. AT21-A and AT22-A; Alpha Diagnostic International, TX, USA). The membrane was washed and then incubated in 1:5000 diluted IgG secondary antibody labeled with HRP for 1h. Using ECL Plus Western Blotting Reagent (Amersham Biosciences) specific bands were detected. The same blots were subsequently stripped and re-probed for actin (Cat. No. MAB1501; Millipore, MA, USA). Densitometry analysis was performed for each experiment. Mean band intensity values were determined using the Kodak^® 1D Image Analysis Software (Eastman Kodak Company). Total protein values were normalized to actin and compared to the vector control samples.

All antibodies with the exception of protein phosphatase 2A catalytic subunit (PP2A/c) (Calbiochem) and beta-actin (Sigma) were purchased from Cell Signaling Technology. Briefly, crushed frozen brains were added to a lysis buffer containing 2% SDS, 60 mM Tris-HCl buffer (pH 6.8), 50 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1mM PMSF, 1 μM leupeptin and homogenized on ice. Homogenates were then sonicated for 10–15 seconds and boiled for 5 minutes at 95°C. Samples were then centrifuged at 15,000xg for 10 min at 4°C. 30 μg of protein were electrophoresed in a SDS–polyacrylamide gel and transferred onto a PVDF membrane (Invitrogen). The membrane was incubated with a blocking buffer (5% nonfat dried milk in PBS containing 0.05% Tween 20 (PBST)) for 1hr at room temperature, followed by incubation with a primary antibody for 1hr at room temperature or overnight at 4°C. The primary antibodies were used 1:1000 dilutions, unless stated otherwise. The membrane was then washed four times in PBST, probed with the secondary HRP-linked antibody (1:5000) in blocking buffer for 1hr at room temperature. Detection of the signal was performed using ECL Plus Western blotting reagents (Amersham Pharmacia Biotech) or Immobilon Western (Millipore). A t-test was used for statistical analysis and a p value of < 0.05 was considered significant.