Anti gfp magnetic beads

Constructs encoding GFP-tagged OsMKK6 or GFP alone were transiently co-expressed with OsMPK4-FLAG in N. benthamiana leaves. Total proteins were extracted from the samples with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% [v/v] glycerol, 1% [v/v] Triton X-100, 1 mM PMSF, 20 mM MG132, and one tablet protease cocktail per 15 mL), and the immunoprecipitated proteins were incubated with anti-GFP magnetic beads (ABclonal, WuHan, China AE079). The immunoprecipitated proteins were separated by SDS-PAGE (12% gel) and analyzed by immunoblotting using anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA, F1804) or anti-GFP antibodies (Sigma-Aldrich, USA; SAB4701015). Following incubation with the corresponding secondary antibody (anti-mouse IgG HRP-linked antibody [CST, Danvers, MA, USA; 7076V] and goat anti-mouse HRP antibody [Licor, Lincoln, Nebraska, USA; 926-80011]) for 1 h, the immunoblot signal was visualized using Immobilon Western HRP substrate (Merck Millipore, Darmstadt, Schwartzwald, Germany; WBKLS0100).

Two breast cancer cell lines T47D and MDA-MB-231 were purchased from ATCC, and cultured in DMEM medium. To stably knock down RUNX1-IT1, two shRNAs targeting RUNX1-IT1 (#1: 5′-TCGAAGACATCGGCAGAAA-3′; #2: ACCACTCCACTGCCTTTAA; Negative control (NC): TTCTCCGAACGTGTCACGT) were inserted into pLKO.1-puro lentiviral vector, followed by infection into T47D and MDA-MB-231 cells. The stable clone was screened by puromycin for 1 week. GPX4 vector was purchased from OriGene (#RC208065) and transfected into T47D and MDA-MB-231 cells to overexpress GPX4. Besides, the full-length coding sequences (CDS) of IGF2BP1 and deletion of KH3/4 mutant were cloned into pEGFP-N1 vector, followed by transfection into HEK293T cells to overexpress IGF2BP1 and its mutant. Protein affinity purification was conducted by using Anti-GFP Magnetic Beads (#AE079, ABclonal, MA, USA) as per the manufacturer’s instructions. Cell transfection was carried out using Lipofectamine 3000 (Thermo Fisher Scientific, CA, USA).