Escherichia coli strain bl21 de3

Protein expression and purification were done using Escherichia coli strain BL21 (DE3; Transgene). Transformed E. coli BL21 clones were grown at 37 °C in LB medium with 50 μg/ml Kanamycin (Beyotime) to OD₆₀₀ of 0.6–0.8. Isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma Aldrich) was added to a final concentration of 0.4 mM to induce protein expression at 16 °C for 16 h. Bacteria were lysed in lysis buffer (20 mM Tris pH 7.8, 500 mM NaCl, 5% glycerol (Sigma Aldrich), 3 mM β-ME (Sigma Aldrich), and 10 mM imidazole (Sigma Aldrich)) with sonication, and centrifuged at 18,000×g for 45 min at 4 °C. The supernatant was purified using Ni-IDA beads resin (ProbeGene). Briefly, the resin was equilibrated with buffer A (20 mM Tris, pH 7.8, 500 mM NaCl, 5% w/v glycerol, and 10 mM imidazole). The supernatant was incubated with Ni-IDA beads at 4 °C for 30 min with rotation. After centrifugation, the resin was the washed with buffer A for three times, and eluted with buffer B (buffer A with 300 mM imidazole). Protein was desalted and concentrated with a 30 K molecular weight cutoff (MWCO) protein concentrator (Pierce), quantified and aliquoted.

The complete ORF I215L, lacking the stop codon, was cloned into the pET-30a(+) vector, and the accuracy of the inserts was verified by DNA sequencing. The confirmed recombinant plasmids were then transformed into Escherichia coli strain BL21(DE3) (TransGen Biotech Co., Ltd., Beijing, China) and grown in Luria-Bertani (LB) medium supplemented with 50 μg/mL kanamycin at 37°C. Once the optical density at 600 nm (OD₆₀₀) value reached 0.6, protein expression was induced by adding 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) for an additional 5 h at 37°C. Subsequent purification procedures were performed as described previously (38 (link)). Purified His-tagged ASFV pI215L (4 mg) was then used to prepare the anti-pI215L mouse polyclonal antibody by the Laboratory Animal Center, Wuhan Institute of Virology, Chinese Academy of Sciences.

HEK‐293T (293T) cells, human normal cervical epithelial cell line (H8), human cervical cancer cell lines (HeLa, C33A and SiHa) and Cisplatin‐resistant cell line HeLa/diamminedichloroplatinum (DDP) were purchased from the Chinese Academy of Science (Shanghai, China). All cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum in a humidified atmosphere of 5% CO₂ at 37 °C. The anti‐Flag M2 agarose and monoclonal mouse antibody against Flag were obtained from Sigma‐Aldrich (St Louis, MO, USA). The polyclonal rabbit antibody against HMGA2 was purchased from the Abcam (Cambridge, MA, USA). The polyclonal rabbit antibodies against GFP, CSNK2A1, Bcl‐2, Bax and β‐actin were purchased from the Proteintech (Wuhan, China). Phospho‐Ser/Thr antibody was acquired from Cell Signaling Technology (Beverly, MA, USA). Cisplatin was obtained from MedChemExpress (Monmouth Junction, NJ, USA). The Escherichia coli strain BL21 (DE3) was purchased from TransGen (Beijing, China). Glutathione Sepharose 4B was purchased from GE Healthcare (Princeton, NJ, USA). CX‐4945 (small‐molecule CK2 inhibitor) was purchased from Selleckchem (Houston, TX, USA).