Anti p65 antibody

Both non-transfected and transfected MCs were fixed with 4% paraformaldehyde for 30 min at room temperature and then permeabilized in PBS containing 0.1% Triton X-100 on ice for 10 min. And then samples were confined by 3% goat serum (Beyotime, Nantong, China) for 1 h at room temperature and incubated by overnight at 4 °C using anti-p50 antibody (Abcam, 1:100), anti-p65 antibody (Abcam, 1:100), anti-NLRP3 inflammasome antibody (Sangon Bio Tech, 1:50), anti-mcp-1 antibody (Sangon Bio Tech, 1:50), anti-TNF-α antibody (Bioworld Tech, Minnesote, CA, USA, 1:50), anti-IL-1β antibody (Bioworld Tech, 1:50), anti-Fn antibody (Sangon Bio Tech, 1:50) or anti-Col4 antibody (Proteintech, Wuhan, China, 1:50). Then, samples were incubated with dylight488 or dylight549 for fluorescent labeling of goat anti-rabbit antibodies (Thermo Fisher Scientific) for 1 h at 37 °C. Subsequently, cells were stained with DAPI for 10 min at room temperature. Finally, the images were observed with confocal microscope and analyzed with LAS AF Lite (Leica).

After the completion of treatment, chondrocytes were fixed, permeabilized, and blocked for 1 h. Then, cells were probed with anti-p65 antibody (Abcam) overnight, followed by an incubation with ant-rabbit Alexa Fluor 546 secondary antibodies for 2 h. Cells were then mounted with DAPI and visualized by Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan).

Proteins were subjected to SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The following primary antibodies were used: (1) an anti-dysbindin antibody [19 (link)], (2) a monoclonal anti-GFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), (3) an anti-Ub antibody (Santa Cruz Biotechnology), (4) an anti-HA antibody (Santa Cruz Biotechnology), (5) an anti-Flag antibody (Sigma), (6) an anti-p65 antibody (Abcam), (7) an anti-Histone H2B antibody (Abcam) and (8) a monoclonal anti-GAPDH antibody (Millipore). Anti-mouse IgG-HRP and anti-rabbit IgG-HRP antibodies (GE Healthcare, UK) were used as secondary antibodies. The proteins were visualized using a SuperSignal West Pico instrument (Thermo Scientific, Waltham, MA, USA).

RAW267.4 cells were incubated on 24 mm × 24 mm coverslips in six-well plates at a density of 1 × 10⁵/well to observe their morphology and P65 nucleation. The culture conditions were the same as those applied to the BLANK, LPS, and LPS + KN-17 treatments for RAW264.7 macrophages. On days 1 and 3, the cells were placed in 4% (v/v) paraformaldehyde (PFA) and their membranes were ruptured with 0.25% (w/v) Triton X-100 for 3–5 min. Each cell group was then blocked with 1% (v/v) bovine serum albumin (BSA) for 30 min. P65 was stained with anti-P65 antibody (Abcam, Cambridge, UK) and goat anti-mouse immunoglobulin G (IgG) secondary antibody (Alexa Fluor 488; Thermo Fisher Scientific). Tetramethylrhodamine (TRITC-rhodamine; Thermo Fisher Scientific) and 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) were used to stain the cytoskeletons and nuclei, respectively. The stained coverslips were fixed on slides with sealant and kept in the dark. CLSM (Carl Zeiss AG, Baden Württemberg, Germany) was used to capture images of the cells under different light sources [23 (link)].