Protein a agarose

CO-IP analysis was performed to detect the ubiquitination level of SLC7A11. Total protein was extracted and quantified according to the method described in Sect. 2.6. 5 µL Protein A agarose and 5µL Protein G agarose (Beyotime, China) were added to the cell lysate and incubated at 4℃ for 1 h. Then, after centrifuging at 12,000 g, 4℃ for 1 min, 2 µg antibody, and the obtained supernatants were incubated overnight at 4℃, while using non-specific immune homologous IgG antibody as the control. The next day, the sediment was cleaned using 0.5 ml 1× wash buffer five times. The sediment was suspended in 30 µL of 1×SDS-PAGE electrophoresis sample buffer and then centrifuged for 30 s to allow the beads and liquid attached to the tube wall to reach the bottom of the tube. Then, The sample was placed at 100℃ for 5 min and centrifuged instantaneously at 14,000 g for 1 min. Finally, the collected supernatant was used for CO-IP. Western blot analysis was performed using the SLC7A11 antibody, and ubiquitination of SLC7A11 was detected using an anti-Ub antibody (Proteintech, China).

rCRD was suspended in phosphate-buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO4, pH 7.4), and injected into 6-week-old mice to generate polyclonal antibodies, following the procedure outlined by Pan et al. [13 (link)]. The IgG of the rCRD antibody was purified using protein A agarose (Beyotime, China). The specificity of the antibody was detected by Western blot analysis [14 (link)].

A codon optimized Tt-tubulin sequence (XP_001023006) for Escherichia coli expression was synthesized by Generay Biotech Co., Ltd. (Shanghai) and then cloned into the pET32a-ΔTRX expression vector (prepared by this lab) to generate pET32a-ΔTRX-Tt-tubulin. The pET32a-ΔTRX-Tt-tubulin plasmid was isolated and transformed into E. coli BL21(DE3) cells. The positive bacterial cells were induced with 1 mM isopropyl b-d-1-thiogalactopyranoside (IPTG). Recombinant Tt-tubulin protein (rTt-tubulin) was purified using a nickel nitrilotriacetic acid column (Ni-NTA; Qiagen, Germany).
The purified rTt-tubulin was emulsified with FCA, and 1 mg of protein was injected into New Zealand white rabbits (weighing approximately 1.3 kg). The animals were then boosted with 0.5 mg of rTt-tubulin in FIA on two separate occasions. Serum was then prepared, and the polyclonal antibody (pAb) titers were determined by enzyme-linked immunosorbent assays (ELISAs) using rTt-tubulin as the antigen. Rabbit immunoglobulin G (IgG) was purified from rabbit antiserum using protein A agarose (Beyotime, Haimen, Jiangsu, China) according to the manufacturer’s instructions.

To detect the interaction among p53, SIRT6 and PARP1, about 6 μg of p53 antibodies was firstly added to 1 ml cell lysate. According to the manufacturer’s protocol, the mixtures were mixed on a rocker at ambient temperature for 2 h. The immunocomplexes were captured by the addition of protein A-agarose (Beyotime, Shanghai, China) mixed at 1:10 ratio, followed by incubation at ambient temperature for 1 h. The beads were washed 5 times and then collected by centrifugation at 12,000 rpm for 30 s. After the final wash, the beads were mixed with 20 μl of 2× loading buffer, heated at 95 °C for 10 min, and analyzed by Western blotting using p53 and PARP1 antibody.

ChIP experiments were conducted with the ChIP Assay Kit (P2078, Beyotime, Shanghai, China) according to the manufacturer's protocol. 1 × 10⁶ RPMI 8226 cells was fixed with 1% formaldehyde, re-suspended by a lysis buffer, and cleaved by ultrasound to shear the DNA into a range of 500–1,000 bp. The DNA and protein complex were then immunoprecipitated with anti-KIF22 antibody (Cat No:13403-1-AP) for 4 h at a room temperature, and the complex was enriched using protein A agarose (Beyotime Institute of Biotechnology). Magnetic beads were isolated and washed. Isolated DNA was purified using a DNA Purification Kit (D0033, Beyotime, Shanghai, China) Precipitated genomic DNA was amplified by RT-qPCR with the CDC25C promoter primers as follows:
forward, 5′- GGCACGAGAAAGAAGCGAAGA-3′,
reverse, 5′- CCGCCAGCCCAGTAACCTA -3′.

Cultured cardiomyocytes were homogenized and lysed in a buffer containing RIPA buffer (Beyotime) and protease inhibitor cocktail (Roche, Basel, Switzerland). Total cell lysates were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatant was collected for further lysis. Then, 30 μl Protein A agarose (P2009, Beyotime, Shanghai, China) beads were added and shaken at 4°C for 4 h to remove nonspecific complex proteins. Anti‐LRP5 (2 µg, 5731S, Cell Signaling Technology, Danvers, Massachusetts, USA ), anti‐AKT (2 µg, ab8805, Abcam, Cambridge, United Kingdom) and normal rabbit IgG (2 µg, 03‐241, Millipore, Burlington, Massachusetts, USA), which was used as a negative control, were added to lysates and incubated overnight. The next day, 30 μl Protein A agarose beads were added to capture the antigen–antibody complex at 4°C for 4 h, and the immunoprecipitates were subjected to Western blotting as described above.