Anti acan

The cells or tissues were lysed, and the total protein concentration was measured using the BCA assay. Equal quantities of proteins were resolved using SDS-PAGE and then transferred onto a PVDF membrane (Merck Millipore, Billerica, MA, USA). Next, the membrane was blocked at 37 °C for 2 h and then incubated with the primary antibody (anti-COLII, anti-ACAN, anti-NF-κB, anti-IFN-β, anti-GAPDH [all from Abcam, Cambridge, MA, USA], or anti-Trim14 [Novus Biologicals] antibody) at 4 °C overnight. The membrane was washed three times with PBST (PBS with Tween 20, Solarbio) and then incubated with the horseradish-peroxidase-labeled anti-mouse or anti-rabbit IgG secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) (diluted 1: 10,000 with PBST) at 37 °C for 2 h. Finally, the membrane was washed five times with PBST and developed using chemiluminescence (BeyoECL Star [Beyotime, Shanghai, China]). The results were analyzed using the ImageJ software (NIH, Bethesda, MA, USA).

IHC staining (Boster Biological Technology, China) was performed according to the manufacturer's protocol to further evaluate the matrix proteoglycan complex deposition and expression in NP tissues. Briefly, the harvested IVD samples were fixed with 4% paraformaldehyde for 48 hours, and then were decalcified in 10% EDTA for 4 weeks. Afterwards, the treated samples were sequentially fixed with paraformaldehyde for 24 h, embedded in paraffin and sectioned at 4 mm. The sections were treated with 3% H₂O₂ for 15 min at room temperature to eliminate endogenous peroxidase activity and incubated with 0.12% trypsin for 30 min at 37 °C to retrieve the antigen, before being blocked with normal goat serum for 15 min at room temperature. Next, the sections were incubated with primary antibodies (anti-ACAN (1:100; Abcam, USA), and anti-Col2 (1:100; Abcam, USA)) overnight at 4°C and then incubated with goat anti-rabbit IgG-HRP secondary antibody. The stained sections were photographed under a microscope (Olympus, Japan). Finally, the sections were counterstained with Harris's haematoxylin and imaged under an optical microscope. The average optical density (AOD) of five randomly selected visual fields (per immunohistochemical slice) was measured using the ImageJ analysis system.

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).