Anti ras

Protein extracts were prepared using high-salt lysis buffer (Hepes 50 mM, pH 7.5, NaCl 500 mM, DTT 1 mM, EDTA 1 mM, 0.1% NP-40) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). Then, 10–20 μg cell and tissues lysates were separated on 10% NuPAGE gels (Invitrogen), transferred onto a nitrocellulose membrane (Invitrogen) and incubated with the following antibodies: anti-p65BTK (BN49); anti-BTK (sc-1696) anti-hnRNPK (sc-25373) from Santa Cruz Biotechnologies; anti-ERK (#9101), anti-phospho-ERK (Thr202/Tyr204) (#4370), anti-eIF4G2 (#5169) from Cell Signaling; anti-actin (A1978), anti-vinculin (V9264), anti-phospho-hnRNPK (SAB4504229) from Sigma-Aldrich; and anti-RAS (#05-516) from Millipore. Each single blot was reprobed with anti-actin or anti-vinculin as loading control. Images were acquired using G:BOX XT4 Chemiluminescence and Fluorescence Imaging System (Syngene, Cambridge, UK) and processed with Adobe Photoshop.

Briefly, tissue from the treated skin of mice or tumor was snap frozen in liquid nitrogen. Tissue was homogenized in lysis buffer, and supernatants were quantified for protein amount. 160 ug of cell lysate was loaded in 10% SDS-PAGE, transferred to a nitrocellulose membrane and incubated with primary antibody overnight at 4°C (1000x dilution for anti-pERK1/2 (Santa Cruz), 2500x anti-GAPDH, 5000x anti-mouse IgG-HRP (Santa Cruz). To detect 1000x anti-RAS (Millipore), lysates were loaded in a 15% gel.

30 μg protein extracts from lung tumors isolated in the three experimental groups or CRISPR-modified KPB6 cells were electrophoresed and immunoblotted using the following primary antibodies and dilutions: anti-SOS1 (Cat#610096, 1:500, BD), anti-SOS2 (Cat#sc-15358, 1:500, Santa Cruz Biotechnology), anti-tubulin (Cat#T5293, 1:10,000, Sigma) and anti-vinculin (Cat#26520-1-AP, 1:5000, ProteinTech) and the corresponding secondary antibodies: Goat anti-mouse DyLight^TM 800 (Cat#SA5-35521, 1:10,000, ThermoFisher Scientific) and Goat anti-rabbit Fluor® 680 (Cat#A21076, 1:5000, Invitrogen). To determine the levels of the GTP-bound levels of RAS in lung tumors (anti-RAS, Cat# 05-516, 1:1000, Millipore), we performed pull-down assays for active RAS as previously reported²⁷ .

Full‐length OTUB1 expression constructs were purchased from ORIGENE. Point mutations to generate OTUB1 C91S were obtained by QuickChange site‐directed mutagenesis PCR (Stratagene, La Jolla). Lentiviral pLA‐CMV‐N‐Flag or pLA‐CMV‐N‐HA vectors were used to generate Flag/HA‐tagged constructs. The pMT107–6×His–ubiquitin plasmid was a generous gift from Dr. Bohmann (University of Rochester, USA). pLKO.1‐puro shGFP and pLKO.1‐puro vectors containing shRNAs targeting OTUB1 (pLKO.1‐shOTUB1_1 (TRCN0000004211), pLKO.1‐shOTUB1_2 (TRCN0000004213), pLKO.1‐shOTUB1_3 (TRCN0000004215)) were purchased from Sigma‐Aldrich.
The antibodies used: mouse monoclonal anti‐FLAG (Sigma‐Aldrich, M2), anti‐RAS (Millipore, Clone 10), anti‐vinculin (Sigma‐Aldrich, clone hVIN‐1), anti‐GAPDH (Sigma‐Aldrich, GAPDH‐71.1), anti‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, 3A7, #9107), anti‐AKT (Cell Signaling, 40D4); rat monoclonal anti‐HA (Roche, 3F10); rabbit monoclonal anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (IHC) (Cell Signaling, D13.14.4E, #4370), anti‐Ki67 (Thermo Scientific #RM‐9106‐S, clone SP6); rabbit polyclonal anti‐DYKDDDDK (Cell Signaling), anti‐OTUB1 (Bethyl Laboratories), anti‐OTUB1 (IHC) (Sigma‐Aldrich, HPA039176), anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, #9101), anti‐pAKT (Cell Signaling, D9E), anti‐RABEX5 (Sigma‐Aldrich).