Ago2 antibody

RIP assay was carried out with the Magna RIP kit (Millipore) in light of the manufacturer’s instructions. BC cells were lysed in lysis buffer and then incubated with immunoprecipitation buffer containing magnetic beads coated with AGO2 antibody (#10686-1-AP, Thermo Fisher Scientific) or IgG antibody (#PP64B, Millipore), and IgG was used as a control. After RNA extraction, the level of circYY1 was detected by qRT-PCR.

A498 and SW839 cells were subjected to RIP lysis buffer (Thermo Fisher Scientific), and the lysate was collected and incubated with the RIP buffer containing magnetic beads coated with Ago2 antibody (Thermo Fisher Scientific) or IgG antibody (negative control; Thermo Fisher Scientific) at 4°C for 4 h. Subsequently, the immunoprecipitated RNAs were isolated and used for qRT-PCR.

The RIP assay was performed using the EZ-Magna RIP Kit (MilliporeSigma) following the manufacturer's recommendations. In brief, C28/I2 cells were lysed in RIP lysis buffer (MilliporeSigma) containing protease inhibitor cocktail and RNase inhibitor for 5 min of incubation on ice followed by centrifugation at 10,000 x g for 10 min at 4˚C. Cell lysate was incubated with RIP buffer containing Protein G magnetic beads (cat. no. 88848; Thermo Fisher Scientific, Inc.) coated with Ago2 antibody (1:50; cat. no. ab186733; Abcam) or the control IgG antibody (1:1,000; cat. no. ab6702; Abcam) overnight at 4˚C according to the manufacturer's recommendations. Next, the magnetic beads were washed three times using RIP wash buffer and treated with proteinase K (Thermo Fisher Scientific, Inc.) at 55˚C for 30 min. Finally, the co-precipitated RNAs were measured using RT-qPCR.