Anti immunoglobulin g anti igg

RIP assay was conducted using Magna RIP^TM RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Transfected SNU-387 and Huh7 cells were lysed in RIP buffer and cell lysates were maintained with magnetic beads conjugated with Anti-Argonaute2 (Anti-Ago2; Abcam) or Anti-immunoglobulin G (Anti-IgG; Abcam). Then, the samples were interacted with proteinase K (Solarbio) and immunoprecipitated RNAs were isolated. The RNAs were extracted and the levels of circ_0000517 and miR-1296-5p were measured via qRT-PCR.

The RIP assay was performed using an EZ-Magna RIP Kit (EMD Millipore) according to the manufacturer's instructions. NP cell lysates were lysed with RIPA buffer (Beyotime Institute of Biotechnology) for 5 min at 4°C. Antibodies including anti-Argonaute 2 (anti-Ago2; cat. no. ab186733; 1:50; Abcam) and anti-Immunoglobulin G (anti-IgG; cat. no. 12-370; 1:100; EMD Millipore) were incubated with protein A/G magnetic beads (Pierce; Thermo Fisher Scientific, Inc.) for 1 h at 4°C. Then, cell lysate was mixed with the beads to incubate for 4 h at 4°C. Beads were washed twice using PBS buffer (Sangon Biotech Co., Ltd.), and the mixture was centrifuged at 2,500 × g for 10 min at 4°C. RNA was purified with 150 µl proteinase K buffer (Roche Diagnostics) and extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and the immunoprecipitated RNA was detected via RT-qPCR as aforementioned.

A Magna RNA immunoprecipitation kit (Millipore) was adopted for RIP assay to confirm the associated relation between circ_0000463 and miR‐924. NSCLC cells were introduced with miR‐924 mimics for 48 h and then cells were disrupted using ice‐cold radio‐Immunoprecipitation Assay (RIPA) lysis buffer. Cell extracts were mixed with anti‐Argonaute 2 (Anti‐Ago2; Abcam) or anti‐Immunoglobulin G (Anti‐IgG; Abcam) for 4 h at 4℃. Then, the magnetic protein A/G beads were added to incubate for 2 h. Beads were washed with PBS thrice, and RT‐qPCR was applied to measure RNA enrichment.