Co ip kit

Co‐IP assay was performed to investigate the interaction protein using Co‐IP kit (Absin, Shanghai, China) according to manufacturer's instructions. Specifically, ESCC cells were fully lysed with 1 mL Co‐IP lysate at 4°C for 30 min, and then the total protein extracted was adjusted to the same concentration. In this study, 1–5 μg of ACLY or SIRT2 antibody was added to 500 μL protein mixture (containing 200–1000 μg of total protein), and cognate IgG was used as a control. The samples were rotated at 4°C overnight. Then, 5 μL protein A and 5 μL protein G were added to incubate at 4°C for 1 h, centrifuged at 12,000 g for 1 min, and the precipitate was washed three times using wash buffer. Then, the mixtures were centrifuged at 12,000 g for 1 min, and the precipitate was retained and resuspended with 20–40 μL of 1 × SDS sample buffer. Finally, the samples were heated at 100°C for 5 min, centrifuged at 14,000 g for 1 min, and the supernatant was used for Western blot assay.

Co-immunoprecipitation (Co-IP) experiments were performed using the Co-IP kit (absin, Shanghai, China) according to the manufacturer's protocol. Anti-flag antibodies and anti-p53 antibodies were used to form an immune complex with the target in the cell lysate. After incubation with protein A, and G for 12 h, the immunoprecipitates were analyzed by western blotting.

The Co-IP kit was purchased from Absin (Cat. No. abs955, China). Experiments were performed following the protocol. The mixture was then detected by Western blotting. The primary antibodies used were: anti-p21 (Cat. No. 2947 S, CST, USA), anti-CDK2 (Cat. No. 2546 T, CST, USA), anti-CDK4 (Cat. No. 12790 T, CST, USA), and anti-CDK6 (Cat. No. 3136 T, CST, USA).

BCA assay kit (KeyGen Biotech, China) was used to quantify the concentration of proteins that were extracted from hiPSC-CMs using lysis buffer. Electrophoresis was used to separate protein samples, which were then transferred to PVDF membranes (Millipore Sigma, China). The primary antibodies were incubated overnight at 4 °C after the membranes had been blocked with skim milk. An ECL imaging system (Bio-Rad, USA) was used to visualize the primary antibodies after they had been conjugated with HRP-conjugated IgG secondary antibody.
Co-IP assays were performed according to the manufacturer’s instructions of the Co-IP kit (Absin, China). Primary antibody (2.5 μg) and cell lysates were mixed overnight at 4 °C. The cell lysates were subsequently mixed with Protein A and Protein G at 4 °C for 3 h. The samples were then centrifuged and washed, and the precipitate was retained. Finally, 30 µl of 1× SDS buffer was added and then boiled, and the supernatant was collected for western blotting after centrifugation.

A Co-IP assay was conducted using a Co-IP kit (# abs955, Absin^®, Shanghai, China) in accordance with the manufacturer's instructions. In brief, RAW 264.7 cells were cultured in dishes of 10 cm in diameter. The cells (1 × 10⁷) were treated with different concentrations of PBD-2 (1, 10, 30, and 50 μg/ml) for 16 h and lysed using a lysis buffer that contained protease inhibitors for 16 h. The soluble faction of the cell lysate was collected after centrifugation at 14,000 × g at 4°C for 10 min. Afterwards, 2 μl of PBD-2 antisera (with titer 1:500,000) or 2 μl of mouse IgG antisera (# sc-2025, Santa Cruz^®, CA, USA) as control were incubated with 200 μg of the cell lysate at 4°C for 12 h. Protein A/G beads (5 μl) were added and incubated at 4°C for 12 h. The precipitates with the immune complexes were collected by centrifugation at 12,000 × g at 4°C for 1 min. The beads were washed with a wash buffer. TLR4 in the precipitates was detected with Western blot by using an antibody against TLR4 (# sc-293072, Santa Cruz^®, Santa Cruz, CA, USA).

Macrophages were cultured in DMEM with 10% FCS in 6-well plates. Cells were treated with IL-4 (100 nmol/L) or LPS (100 ng/mL) for the indicated times. After stimulation, cells were washed once in cold PBS and lysed in RIPA buffer (50 mmol/L Tris–HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA pH 7.4) with protease and phosphatase inhibitor cocktails (Sigma) for 10 min on a rocker at 4 °C. Protein concentration was determined using a BCA assay. Protein samples were analysed by SDS polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto PVDF membranes (Millipore, CA)60. Each polyvinylidene fluoride membrane was blocked with TBST (100 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 0.05% Tween 20) with 5% BSA for 1 h and then incubated with primary antibodies overnight on a shaker at 4 °C. The appropriate HRP-coupled secondary antibody was then added and detected through chemiluminescence (Millipore). GAPDH and β-tubulin were used as protein loading controls. Coimmunoprecipitation was performed with indicated antibodies and G/A beads following information of co-IP kit (absin, abs955). Quantitative analyses with a p-value between indicated groups in the WB were calculated via ImageJ.