Ras-GTP Extraction and Western Blot Analysis

PVN samples were homogenized in a lysis buffer and Western blot was performed as previously described (Su Z. et al., 2014 (link)). Specifically, Ras active protein (Ras-GTP) was extracted from total tissue lysates by adding 80 ug GST-Raf1-RBD per 500 ug sample and using Active Ras Pull-Down and Detection Kit (Thermo scientific, USA) and assessed by western blot immediately or stored at −20°C.
Primary antibodies used included the following: anti-AT1-R (1:200, Millipore, USA), anti-p-ERK1/2 (1:300, Cell Signaling Technology, USA), anti-p-p38 (1:500, Cell Signaling Technology, USA), anti-p38 (1:1000, Cell Signaling Technology, USA), anti-Bax (1:500, Santa Cruz Biotechnology), anti-Bcl-2 (1:200, Santa Cruz Biotechnology), anti-Cleaved caspase-3 (1:600, Cell Signaling Technology), anti-ERK1/2 (1:1000, Cell Signaling Technology, USA), anti-Ras (1:200, Abcam Cambridge, UK), anti-β-actin (1:5000, Cell Signaling Technology), anti-GAPDH (1:1000, Cell Signaling Technology). The levels of the above proteins were determined as previously described (Su Z. et al., 2014 (link)). AT1-R protein content was normalized to GAPDH amounts. Ras, p-ERK1/2, and p-p38 levels were normalized to total Ras, ERK1/2 and p38, respectively. Bax, Bcl-2 and cleaved caspase-3 protein levels were normalized to β-actin.