Anti igg antibodies

The human embryonic kidney 293T (HEK293T) and human liver cancer HepG2 cells were purchased from American type culture collection (ATCC, Maryland, MD, USA). Both cell lines were cultured in high glucose Dulbecco’s Modified Eagle’s Medium, supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Carlsbad, CA, USA) and penicillin (100 U/mL)/streptomycin (100 mg/mL), at 37°C in 5% CO₂. β-actin, CD63, CD9, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), CCND2, CCNE2, cell division protein kinase 6 (CDK6), and anti-IgG antibodies were purchased from Abcam (Cambridge, MA, USA), and an Apo-A1 and anti-IgG antibody were purchased from R&D (Minneapolis, MN, USA). Cy5-labeled miR-26a was synthesized by Integrated DNA Technologies (Coralville, IA, USA). Other reagents were purchased from Thermo Fisher Scientific. The FBS was microvesicle depleted by 2 h ultracentrifugation at 100,000× g, followed by filtrating with a 0.22 μm steritop filter (Millipore, Billerica, MA, USA).

RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (MilliporeSigma). SRA01/04 cells were lysed in RIP lysis buffer (MilliporeSigma). The obtained cell lysate (100 µl) was centrifuged at 40,000 × g at 4°C for 10 min and incubated with 50 µl A/G magnetic beads conjugated with 5 µg anti-AGO₂ (5 µg; cat. no. ab32381; Abcam) or 5 µg anti-IgG antibodies (cat. no. ab172730; Abcam) for 1 h at 4°C. Subsequently, the beads were washed three times using RIP Wash Buffer. The beads were then incubated with proteinase K buffer at 55°C for 30 min to digest the protein. Finally, XIST and miR-34a enrichment was measured via RT-qPCR.