Ripa lysis buffer

Total protein was extracted from metastatic liver tissue or cell lines with or without CXCL12 (cat. no. ab259416; 100 ng/ml, Abcam) using RIPA lysis buffer (MedChemExpress, Inc.), according to the manufacturer's protocol. Total protein was quantified using a BCA Protein Quantification kit (Pierce; Thermo Fisher Scientific, Inc.) and proteins (20 µg/lane) were separated via SDS-PAGE on a 10% gel. The separated proteins were subsequently transferred onto NC membranes (EMD Millipore) and blocked with 1% BSA (Thermo Scientific, Inc.) for 1 h at room temperature. The membranes were then incubated with rabbit anti-MTSS1 (cat. no. ab204127; 1:500), anti-Rac (cat. no. ab180683; 1:2,000, anti-CDC42 (cat. no. ab187643; 1:20,000) and anti-GAPDH (cat. no. ab8245; 1:10,000) (all from Abcam) primary antibodies at 4°C overnight. Following primary antibody incubation, the membranes were incubated with a horseradish peroxidase conjugated anti-rabbit secondary antibody (cat. no. ab97051; 1:20,000, Abcam) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence system (Amersham, Cytiva, Inc.), according to the manufacturer's protocol, on an Odyssey scanner (LI-COR Biosciences, Inc.). Densitometric analysis was performed using ImageJ software V1.8.0 (National Institutes of Health).

LUAD cells were lysed using RIPA lysis buffer (MedchemExpress, China). The protein lysate was separated via SDS–PAGE (Invitrogen) and transferred to PVDF membranes (Millipore, USA) for analysis.²¹ The membranes were incubated with anti‐GRAP2 (1:2000 dilution, Cambridge, MA, USA, ab224613) and anti‐GAPDH (1:5000 dilution, Proteintech, China, 60,004‐1‐Ig) antibodies at 4°C overnight and then with HRP‐labeled secondary antibody (1:2000 dilution, Beyotime, China, A0181) at room temperature for 2 h. Western blotting results were analyzed with ImageJ software.

Western blot analysis was performed using standard methods (16 (link)). Proteins were extracted from tissues and cells using RIPA Lysis Buffer (MedChemExpress). Briefly, 50 µg protein/lane was separated by SDS-PAGE on 12.5% gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA for 2 h at room temperature and then incubated with primary antibodies (1:2,000) at 4°C overnight. Subsequently, the membranes were incubated with the corresponding secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:10,000; ab6721; Abcam) at room temperature for 1 h and the protein bands were visualized using an enhanced chemiluminescence detection system (Amersham; Cytiva). ImageJ (version 1.8.0; National Institutes of Health) was used for semi-quantification.
NE-PER Nuclear Extraction Reagent (Thermo Fisher Scientific, Inc.) was used to isolate and extract nuclear proteins, respectively. Specific detailed steps were performed as described previously (17 (link)).