Anti n cdh

After rehydration, tissue sections were blocked by goat serum, treated with hyaluronidase (0.8%) for 20 min at 37 °C, and then incubated with primary antibodies (anti-N-CDH (1:500; Proteintech, China), anti-ITGβ (1:1000; Abcam, USA) overnight at 4 °C. After an additional washing step, the sections were incubated with the fluorescent dye-conjugated secondary antibody (1:1000; Proteintech, China) for 2 h at room temperature. The stained sections were then labeled with DAPI and imaged under a laser scanning confocal microscope (Leica, Germany).

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).