1xphosstop

Cells were rinsed with cold PBS and immediately lysed in buffer supplemented with 4xcOmplete™ EDTA-free Ultra Protease Inhibitor Cocktail (Sigma-Aldrich) and 1xPhosSTOP™ (Sigma-Aldrich) at 4 °C for 30 min. Following centrifugation at 14,000×g for 5 min, supernatants were transferred into a clean tube and protein concentrations were determined using the Precision Red Advanced Protein Assay (Cytoskeleton, Inc. ADV02-A). Lysates were diluted and analyzed using the Human Phospho-MAPK Arrays (Proteome Profiler; R&D Systems; Minneapolis, MN, USA) according to the manufacturer’s instructions. Nitrocellulose membranes were scanned using a ChemiDoc™ Imaging Systems (BIO-RAD Laboratories, Inc.).

For the evaluation of Ras activation levels after treatment with deltarasin, gemcitabine, C14 or P8, a RAS-GTP pull-down assay was performed using the Ras Activation Assay Biochem Kit according to the standard procedure (BK008; Cystoskeleton, Inc.). Briefly, 3 × 10⁶ cells were lysed in ice-cold lysis buffer (400 μl) supplemented with complete EDTA-free Ultra Protease Inhibitor Cocktail and 1xPhosSTOP (Sigma-Aldrich). Lysates were centrifuged, the supernatants collected, and the protein concentration determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). 300 μg of total protein were incubated by end-over-end rotation with 100 μg of Raf-RBD–conjugated beads for 1 h, followed by centrifugation at 18.8g for 10 min. The beads were centrifuged at 18.8g for 10 min, rinsed with 500 liters of wash buffer, boiled in 2x Laemmli sample buffer, and then subjected to Western blot analysis using the pan-Ras and KRas (F234 Santa Cruz Biotechnology 1:100) antibody.

Cells were rinsed with cold PBS and immediately lysed in buffer supplemented with 4xcOmplete™ EDTA-free Ultra Protease Inhibitor Cocktail (Sigma-Aldrich) and 1xPhosSTOP™ (Sigma-Aldrich) at 4 °C for 30 min. Following centrifugation at 14,000×g for 5 min, supernatants were transferred into a clean tube and protein concentrations were determined using the Precision Red Advanced Protein Assay (Cytoskeleton, Inc. ADV02-A). Lysates were diluted and analyzed using the Human Phospho-MAPK Arrays (Proteome Profiler; R&D Systems; Minneapolis, MN, USA) according to the manufacturer’s instructions. Nitrocellulose membranes were scanned using a ChemiDoc™ Imaging Systems (BIO-RAD Laboratories, Inc.).