Bca protein assay kit

Radioimmunoprecipitation assay lysis buffer was used to prepare SW480 and SW620 cell lysates. After microcentrifugation, the protein samples were quantified using a BCA Protein Assay kit (Abcam, USA). Protein samples (20 µg) were separated using 10% SDS-PAGE and transferred to a PVDF membrane at a constant voltage of 75 V. The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated with anti-AURKB (Cat#:36-5200; 1:1,000; Thermo Fisher), anti-Bax (Cat#:33-6400; 1:1,000; Thermo Fisher), anti-Bcl-2 (Cat#: MA5-11757; 1:1,000; Thermo Fisher), and anti-GAPDH antibodies (Cat#: MA1-16757; 1:1,000; Thermo Fisher) overnight at 4°C. The next day, the membranes were incubated at room temperature with HRP-conjugated goat anti-rabbit/mouse secondary antibodies (Cat#: SA5-10204 or A32727; 1:1,000; Thermo Fisher) for 2 h. The blots were then developed using BeyoECL Plus mix substrate (Beyotime, USA), and ImageJ (Image J Software, National Institutes of Health, USA) was used to quantify the target proteins.

RIPA buffer containing protease inhibitors was used to extract proteins from rat spinal cord tissues and BV-2 cells. Protein concentrations were then determined using a BCA Protein Assay Kit (Abcam). Protein samples (20 μg/lane) were separated via 10% SDS-PAGE and the resolved proteins were transferred onto PVDF membranes. Membranes were blocked with 5% bovine serum albumin at room temperature. After blocking, membranes were incubated overnight at 4 °C with primary antibodies against TLR4 (1:1000; Abcam), MyD88 (1:1000; Abcam), p65 (NF-κB) (1:1000; Abcam), p-p65 (phospho-NF-κB) (1:1000; Cell Signaling), NLRP3 (1:1000; Abcam), ASC (1:1000; Affinity Biosciences), caspase 1 (1:1000; Affinity Biosciences), N-GSDMD (1:1000; Abcam), and GAPDH (1:1000; Abcam). Thereafter, they were washed three times with Tris-buffered saline Tween-20. Subsequently, an HRP-conjugated IgG secondary antibody (1:5000; Santa Cruz, Waltham, MA, USA) was added and membranes were incubated at room temperature for 1 h. GAPDH was used as the internal reference. An enhanced chemiluminescence detection kit (Thermo Fisher Scientific) was used to detect the bands, which were then quantified using Gel-Pro Analyzer software (version 4.0; Media Cybernetics, Silver Spring, MD, USA).

The NP tissues and the differently treated NP cells were lysed with RIPA buffer (Sangon Biotech, Shanghai, China). Next, the proteins were quantified by a BCA Protein Assay Kit (Abcam, Cambridge, UK), and then the proteins with different molecular weights were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred into polyvinylidene fluoride (Millipore, Massachusetts, USA) membranes. The above membranes were incubated with 5% skim milk for approximately 1 h and further incubated with specific primary antibodies, including anti-CILP (Abcam, 1: 1000 dilution, ab192881) and anti-β-actin (Abcam, 1: 1000 dilution, ab8227) at 4 °C overnight. After being washed about two times, the membranes were further incubated with the secondary antibody (Abcam, 1: 2000 dilution, ab205718) for about 1 h at room temperature. Ultimately, the enhanced chemiluminescence reagents (Millipore, Boston, Massachusetts, USA) and Image J (National Institutes of Health, Bethesda, USA) were applied to observe and analyze all the protein bands.

Cells were pelleted by centrifugation, washed once with ice-cold PBS, and lysed on ice for 30 min using the cell lysis buffer (p0013, Beyotime, Shanghai, China) according to the manufacturer’s extraction protocol. Protein quantitation was performed using the BCA Protein Assay Kit (ab102536, Abcam, Shanghai, China). A total of 30 μg of protein was denatured in Laemli buffer at 95°C for 5 min, and western blotting was performed using the Bio-Rad system (TGX 10–15% gels). Transfer was performed using the Trans Blot turbo system (Bio-Rad) into PVDF membranes. Images were acquired using the Bio-Rad Imaging Chemidoc XPS + system. Secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were purchased from Beyotime (A0208; A0216). Proteins were detected using the following antibodies: PARP1 (66520-1-Ig, Proteintech, Wuhan, China); Caspase 3 (9662, Cell signaling technology, Shanghai, China); P21 (60214-1-Ig, Proteintech); Cyclin D1 (60186-1-Ig, Proteintech); AKT (60203-2-Ig, Proteintech); AKT-phospho-S473 (66444-1-Ig, Proteintech); 4EBP1 (60246-1-Ig, Proteintech); c-MYC (10828-1-AP, Proteintech); Histone H3 (acetyl K9) (ab32129, Abcam); S6 Ribosomal Protein (AF6354, Affinity, Jiangsu, China); Phospho-S6 Ribosomal Protein (Ser235/236) (4858T, Shanghai, China), and Phospho-4E-BP1 (S65) (YP0618, Immunoway, TX, United States).