Active ras pull down and detection kit

Cells were grown in 10-cm dishes, and were (or not) pre-treated with 5 mM N-Acetyl cysteine (NAC) for 2 h before H₂O₂ administration. H₂O₂ was added at the final concentration of 500 μM for 5, 30 or 60 min. After oxidative stimulus cells were harvested and Ras-GTP was identified by precipitation with GST-tagged Raf1-RBD with the Active Ras Pull-Down and Detection Kit (Thermo Scientific) following the manufacturer’s instructions. Briefly, cells were lysed in a buffer containing 25 mM Tris·HCl (pH 7.2), 150 mM NaCl, 5 mM MgCl₂, 1% Nonidet P-40, 5% glycerol, and 1× protease inhibitor mixture (Roche Molecular Biochemicals). Lysates were clarified, the protein concentrations were normalized, and lysates (500 μg each) were incubated for 1 h at 4°C with 80 μg of GST-Raf1-RBD. Then GST beads were washed twice in the wash buffer. Samples were eluted and separated by SDS-PAGE electrophoresis in 15% gels and then transferred to nitrocellulose membranes (GE Healthcare, UK). Immunoblotting analysis was conducted with anti-c-K-Ras (clone 234-4.2) and anti-H-Ras Antibody (clone 7D7.2 MAB3291 Millipore). Moreover, as a positive loading control of the ability of GST-RDB resin to retain the GTP-bound form of Ras proteins, a fraction of the lysates was loaded with a stable non-hydrolysable GTP analog (GTP-γS, guanosine 5′-O-[gamma-thiol] triphosphate) following the manufacturer’s instructions.

HUVECs were washed with ice-cold Tris-buffered saline (TBS; 25 mM Tris-HCl [pH 7.5], 150 mM NaCl), lysed using lysis buffer (25 mM Tris-HCl [pH 7.2], 150 mM NaCl, 5 mM MgCl₂, 1% NP-40, 5% glycerol, and protease inhibitor cocktail), scraped, and collected in a microtube. After a 5-min incubation on ice, cell debris was removed by centrifugation at 16,000g at 4 °C for 15 min. The pull-down assay was conducted using an Active Ras Pull-Down and Detection kit (16117, Thermo Fisher Scientific) according to the manufacturer’s instructions, and proteins were recovered from the resultant immunoprecipitates in 2× SDS sample buffer.

Active Ras GTPase was detected and quantified using an active Ras pull-down and detection kit from Thermo Fisher Scientific (Grand Island, NY) according to the manufacturer’s instruction. Briefly, GST-Raf1-RBD (Ras-binding domain), coupled to glutathione agarose resin, was used to pull down the activated Ras. Addition of GTP or GDP was used as a positive control or negative control. Eluted samples were then separated on a 12% acrylamide gel and analyzed using ani-Ras antibody provided in the kit.