Anti rac1

Cell were lysed with RIPA lysis buffer (C500005, Sangon biotech, China). Total protein (20 μg) was separated on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto a 0.45 μm PVDF membrane (Merck, Germany). The membranes were blocked in 5% nonfat milk solution for 1 h at room temperature and were then incubated with the primary antibody at a 1:1000 dilution overnight at 4°C. Then the goat anti-Rabbit HRP-labeled secondary antibody (#4030-05, SouthernBiotech, Birmingham, USA) was incubated for 2 h at room temperature. The ECL regents (Pierce, Rockford, IL, USA) was used to detect the signal of the target proteins. The specific primary antibodies were purchased from the following resource: anti-LIMD2 (#ab167895, Abcam, Cambridge, UK), anti-FAK(#12,636-1-AP,proteintech, HuBei, China), anti-Phospho-FAK(Tyr397)(#8556 T,CST, Boston, USA), anti-B-tubblin(HRP-66031, Proteintech, HuBei, China), anti-RAC1(#24,072-1-AP,Proteintech, HuBei, China) and Tensin 2 Antibody (#11,990, CST,Boston, USA).

Cells were lysed in a protein solubilization buffer. Protein extracts were prepared with a Total Protein Extraction Kit according to the manufacturer’s instructions (BestBio, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore). β-actin or GAPDH served as a loading control. Primary antibodies included anti-GAPDH, anti-β-actin (ZSGB-BIO, China), anti-TNFAIP8, anti-Rac1 (Proteintech), anti-Flag (Sigma), as well as anti-ERK1/2, anti-p-ERK1/2, anti-MEK, and anti-p-MEK (CST). Protein bands were visualized using a FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences).

The NPCs and NPC spheroids were collected and lysed in lysis buffer (Beyotime, China) containing a mixture of protease inhibitors phenylmethanesulfonyl fluoride (PMSF, Beyotime) and phosphatase inhibitor cock-tail I (Sigma, USA). A BCA protein quantification kit was used to determine the protein concentration of each sample according to the manufacturer's protocol. Depending on the results, equivalent amounts of protein (20 μg) were loaded on the 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis was completed, the separation gel was removed, and the target protein in the gel was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking with the 3% bovine serum albumin (BSA) blocking solution, the membranes were incubated with the primary and secondary antibodies, developed, fixed and exposed. The primary antibodies used in this study were as follows: anti-ACAN (1:1000; Abcam, USA), anti-Col1 (1:1000; Abcam, USA), anti-Col2 (1:1000; Abcam, USA), anti-matrix metallopeptidase-13 (MMP-13; 1:500; Proteintech, China); anti-N-CDH (1:500; Proteintech, China), anti-Integrinβ1 (ITGβ1; 1:1000; Abcam, USA), anti-Filamin A (1:1000; Abcam, USA), anti-Rac1 (1:500; Proteintech, China), anti-p-FAK 1:500; Proteintech, China), anti-Src (1:500; Proteintech, China), and anti-GAPDH (1:500; Proteintech, China).

Antibodies with working dilution, company source, and catalog number are listed as below: anti-CD44 (1:200 for IF or 1:1000 for WB; Abcam, #ab119863), anti-Hermes-1 (1:100; Bioxcell, #BE0262), anti-N-cadherin (1:200 for IF or 1:1000 for WB; BD Bioscience, #610921), anti-β-catenin (1:300 for IF or 1:1500 for WB; BD Biosciences, #610154), anti-E-cadherin (1:300 for IF or 1:1500 for WB; BD Biosciences, #610182), anti-γ-catenin (1:200 for IF or 1:1000 for WB; BD Biosciences, #610253), anti-RhoA (1:200 for IF or 1:1000 for WB; Cytoskeleton, #ARH03), anti-Rac1 (1:1000; Proteintech, #24072-1-AP), anti-pMLC (1:200 for IF or 1:1000 for WB; CST, #3671), anti-NKp46 (1:200 for IF; eBioscience, #11-3351-82). All the secondary antibodies (1:300 for IF and 1:3000 for WB) were purchased from Life technologies. Phalloidin (1:200) and Hoechst (1:2000) were purchased from Invitrogen. Z-ADD-CMK (Merck Millipore, #368050), LysoTracker® Red DND-99 (Invitrogen, #L7528) and Y27632 (MCE, # HY-10071) were purchased and used according to manufacturer’s instructions.