Anti gapdh

Proteins from the tissues and cells were isolated using a protein extraction kit (Beyotime Biotechnology, Haimen, China), and the protein content was determined using a BCA Protein Assay Kit (Beyotime, Jiangsu, China). The protein samples were separated by SDS-PAGE gel electrophoresis (8–15%) and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). After 2 h of blocking with 5% bovine serum albumin in TBST buffer at room temperature, the PVDF membranes were individually incubated overnight at 4℃ with the following primary antibodies: anti-ACOT7 (Abcam, Cambridge, UK) and anti-GAPDH (MultiSciences Biotech Co., Ltd, Hangzhou, China). After washing with TBST, the membranes were incubated at room temperature for 1 h with peroxidase-conjugated goat anti-rabbit immunoglobulin G antibodies (MultiSciences Biotech Co., Ltd) at a 1:2000 dilution. Protein expression was detected by the enhanced chemiluminescence method. Protein bands were imaged using Image Lab software (BIO-RAD, Hercules, CA, USA) and normalized with GAPDH as an internal control. The methods for determining protein content were described previously.26 The experiments were repeated at least three times.

After the indicated treatments, the total protein of IVD cells was extracted using a Western and IP cell lysis kit (Beyotime, China). The protein concentrations were measured using a BCA protein assay kit (Beyotime, China). Equal protein amounts (30 μg) were resolved on 10–12% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 5% nonfat milk in TBST for 2 h at room temperature, the membrane was washed with TBST and incubated overnight at 4 °C with primary antibodies. The following antibodies were used: anti-receptor-interacting protein kinase 1 (RIPK1; 1:1000, Beverly, MA, CST, USA), anti-RIPK3 (1:1000, Abcam, Cambridge, MA, USA), anti-mixed lineage kinase domain-like (MLKL; 1:1000, Abcam, USA), and anti-GAPDH (1:1000, MultiSciences Biotech, China). Subsequently, the membranes were washed with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 60 min at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Thermo, Rockford, IL, USA) as described previously [15] (link)