Polyvinylidene fluoride membranes

Naïve CD4⁺ T cells were lysed in protein extraction solution (Solarbio) plus protease and phosphatase inhibitor (Solarbio). Proteins were quantified using BCA reagent (Servicebio). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out using precast gel (Willget Biotech), and proteins were transferred onto polyvinylidene fluoride membranes (Servicebio). The membranes were incubated with 5% BSA (37°C, 1.5 h), then incubated with PCSK9 (1:500), p‐ERK (1:1500), ERK (1:3000), p‐JNK (1:1500), JNK (1:3000), p‐NF‐κB p65 (1:1500), NF‐κB p65 (1:3000), or GAPDH antibody (1:8000) (All bought from Abcam; 37°C, 1 h). Afterward, membranes were incubated with secondary antibody (1:5000; 37°C, 1 h; Servicebio). The ECL Kit (Servicebio) was used for detection of blots.

Cells and tissues were collected and lysed with RIPA buffer containing phenylmethanesulfonylfluoride (Beyotime, China). Protein concentration was determined with a BCA assay kit (Beyotime, China). Equivalent amounts of total protein were separated by 10% sodium dodecyl sulfate polyacrylamide (servicebio, China) gel electrophoresis and then transferred to polyvinylidene fluoride membranes (servicebio, China). Blocking was performed in TBST containing 5% nonfat milk, and membranes were incubated with primary antibody overnight at 4 °C. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C. An ECL kit (Multisciences, China) was used to detect the proteins. Image J software (NIH, MD, USA) was used to evaluate the relative expression levels of different proteins. Primary antibodies for BRD4 (ab128874), caspase-1 (ab1872), Il-1β (ab7632), cleaved N-terminal GSDMD (ab215203), vimentin (ab92547), caspase-3 (ab2302) and GAPDH (ab9485) were purchased from Abcam. Antibodies for Cl-caspase-1 (4199), E-ca (3195), NLRP3 (15101), NF-κB-p65 (8242), phospho-NF-κB-p65 (3033), IkBa (4812), and p-IkBa (2859) were from Cell Signaling Technology. Goat and anti-rabbit secondary antibodies were purchased from Wuhan Boster Bio-engineering Limited Company, China.

Total proteins were extracted from the nucleus pulposus cells using RIPA buffer (Solarbio, Beijing, China). Protein concentration was determined by BCA Protein Assay Kit (Solarbio, Beijing, China). Equal amount of protein was resolved on a 10% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Servicebio, Wuhan, China). The membranes were then washed with 5% nonfat milk in Tris-buffered saline plus 0.1% Tween 20. Subsequently, the membrane was incubated at 4 °C overnight with antibodies specific for c-Caspase3, c-Caspase7, MMP3, MMP13, Collagen II, Aggrecan, SSA1 and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After the membranes were washed three times with TBST, horseradish peroxidase-conjugated secondary antibody was incubated for 1 h at indoor temperature. The immunoreactivities were detected enhanced chemiluminescence kit (Santa Cruz Biotechnology, Dallas, TX, USA).