Rip buffer

The RIP assay was performed to explore the interaction between ACSM3 and IGF2BP utilizing a Magna RIP RNA Binding Protein Immunoprecipitation kit (EMD Millipore) according to the manufacturer's protocol. HL-60 cells were lysed on ice for 5 min by means of RIP buffer (Beyotime Institute of Biotechnology). The cultivation with anti-IGF2BP2 antibody (cat. no. 11601-1-AP; ProteinTech Group, Inc.) and anti-IgG (cat. no. 30000-0-AP; ProteinTech Group, Inc.) were performed at 37˚C overnight. The protein A/G beads (40 µl; cat. no. 20422; Thermo Fisher Scientific, Inc.) were coated with 2 µg anti-IGF2BP2 2 or 2 µg anti-IgG antibodies at 4˚C for 6 h. Afterwards, the captured protein-RNA complex was digested with 0.5 mg/ml proteinase K containing 0.1% SDS to extract RNA. To remove non-specific adsorption as much as possible, the magnetic beads were repeatedly washed with RIP washing buffer. RT-qPCR was applied for the analysis of resultant RNA levels.

An argonaute 2 (AGO2) RIP assay (25 (link)) was performed to identify the interaction between circRNA_PTPRA and miR-582-3p using a Magna RIP RNA Binding Protein Immunoprecipitation kit (cat. no. 17-701; EMD Millipore) according to the manufacturer's protocol. Cells were lysed using RIP buffer (Beyotime Institute of Biotechnology) on ice for 5 min. The anti-Argonaute 2 (cat. no. ab186733; dilution, 1:50) and anti-IgG (cat. no. ab109489; dilution: 1;300) antibodies were obtained from Abcam and used according to the manufacturers' protocols. The magnetic beads (40 µl) were coated with 2 µg anti-Argonaute 2 or 2 µg anti-IgG antibodies at 4˚C for 6 h. Subsequently, the cell lysate (20 µg protein) was added into the above magnetic beads-antibody mixture and incubated at 4˚C for 1 h, according to the manufacturer's instructions. Resultant RNA levels were analyzed via reverse transcription-quantitative (RT-q)PCR analysis.

The RIP assay is a powerful tool used to assess the binding of RNA molecules to proteins in cells and to assess the dynamic process of post transcriptional regulatory networks (20 (link)). Thyroid cancer cells were transfected with miR-654-3p mimic and mimic control at 37°C for 48 h, and were lysed using RIP buffer (Beyotime Institute of Biotechnology). The magnetic beads were coated with anti-Ago2 (1:50; cat. no. ab186733; Abcam) or IgG (1:300; cat. no. ab109489; Abcam) antibodies and the cell lysate was incubated with the magnetic beads at 4°C for 1 h, according to the manufacturer's instructions.