Ab32417

Total proteins were harvested in ice with RIPA buffer (89,900, Thermo Fisher) supplemented with protease inhibitor cocktail (HY-k0010, MCE) and quantified using BCA assay (P0009, Beyotime). Subsequently, 20 μg of the proteins were electrophoresed by 10% SDS-PAGE (1,610,183, Bio-Rad) and then transferred onto PVDF membranes (IPVH00010, Millipore) using a Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% nonfat milk at room temperature and cut into different strips according to the position of the marker. Then different strips were accordingly incubated with indicated primary antibodies at 4 °C overnight. The next day, membranes were probed with HRP-conjugated secondary antibody (1:5000, 7074P2, CST) at room temperature. Protein bands were visualized using hypersensitive ECL kit (BL523B-2, Biosharp) on a Bio-Rad imaging system and analyzed with ImageJ software. The primary antibodies used were diluted with primary antibody dilution Buffer (P0023A, Beyotime) containing anti-Fibronectin (1:1000, ab32417, Abcam), anti-N-cadherin (1:1000, 13,116, CST), anti-Vimentin (1:1000, 5741, CST), anti-p62/STSQM1 (1:10,000, ab109012, Abcam), and anti-LC3B (1:1000, 3868, CST), anti-GAPDH (1:3000, 5174S, CST).

Isolation of active RAS-GTP was performed using the Active Ras Pull-Down and Detection Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. RAS abundance was measured by Western blot. Western blot analysis of RBD pull-down lysates was performed with mouse anti-KRAS antibody (WH0003845M1, Sigma), rabbit anti-NRAS (ab167136, Abcam), rabbit anti-HRAS (ab32417, Abcam), and mouse anti-GAPDH (sc-4772, Santa Cruz Biotechnology). Input lysates were analyzed with mouse anti-pERK (675502, Biolegend) and rat anti-ERK (686902, Biolegend). All antibodies were diluted to 1/1000 in Licor intercept antibody dilutent (927–66003, Licor).

Co-immunoprecipitation (co-IP) was performed according to the manufacturer's instructions (Pierce Co-Immunoprecipitation Kit, Thermo Scientific). Antibodies against QPCT (sc-517122, Santa Cruz, 1:50) and HRAS (ab32417, Abcam, 1:50) were used.

Total cell and tissue lysates were prepared in 1× sodium dodecyl sulphate (SDS) buffer. Identical quantities of protein were separated by SDS gel electrophoresis and transferred onto nitrocellulose filter membranes. After incubating with antibodies specific for QPCT (ab201172, Abcam, CA, USA) and GAPDH (sc-25778; Santa Cruz, CA, USA), the blots were incubated with IRDye 800-conjugated goat anti-rabbit IgG, and bands were detected using an Odyssey infrared scanner (Li-Cor). GAPDH was used as the loading control. The other antibodies used for western blot were against IRS1 (ab52167, Abcam, CA, USA), NF-κB (p65) (8242, Cell Signalling Technology), HRAS (ab32417, Abcam, CA, USA), CBL (ab32027, Abcam, CA, USA), GAB1 (ab59362, Abcam, CA, USA), NAF1 (ab157106, Abcam, CA, USA), MAPK8 (ab199380, Abcam, CA, USA), MAPK10 (ab126591, Abcam, CA, USA), FAK (PTK2) (ab40794, Abcam, CA, USA), p-FAK (ab81298, Abcam, CA, USA), ERK1/2 (4695, Cell Signaling Technology), p-ERK1/2 (4370, Cell Signaling Technology), AKT (4691, Cell Signaling Technology), p-AKT (4060, Cell Signaling Technology), Stat3 (9139, Cell Signaling Technology), p-Stat3 (9145, Cell Signaling Technology), and ubiquitin (3936, Cell Signaling Technology).