Ultra protease inhibitor cocktail

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 lysed using radioimmunoprecipitation assay buffer (Sigma) containing 1× cOmplete Ultra protease inhibitor cocktail (Sigma). Proteins were separated by SDS-PAGE using 4% to 20% precast Mini-Protean TGX gels (Bio-Rad) before transfer onto a nitrocellulose membrane using the Trans-Blot turbo transfer system (Bio-Rad). Membranes were blocked for 1 h at room temperature (RT) with 5% milk in phosphate-buffered saline (PBS) with 0.1% Tween 20. Membranes were probed using the following primary antibodies for 16 h at 4°C: IFI44 (PA5-65370; ThermoFisher), IFI44L (ARP46166; VWR), and β-actin (ab8227; Abcam). Membranes were washed and probed with anti-IgG horseradish peroxidase-conjugated antibodies (Dako) prior to chemiluminescent detection.

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.