Normal mouse immunoglobulin g

Briefly, an RIP assay using SPC-A1 cells was performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). Cells were transfected with Flag-Ago2 lentivirus (Genechem, Shanghai, China) and then treated with Radio Immunoprecipitation Assay (RIPA) Lysis Buffer. The cell lysates were incubated with antibodies against Ago2 (Millipore) or normal mouse immunoglobulin G (Millipore). After being treated with buffer, the immunoprecipitated RNAs were extracted from the cell lysates, and western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis were then performed.
An RNA pull-down assay was conducted to verify the relationship between hsa_circ_0006571 and miR-138 using the Pierce Magnetic RNA-Protein Pull-Down Kit (BersinBio, Guangzhou, China). For RNA pull-down, specific probes were used to hybridize hsa_circ_0006571, which was pulled down and identified through qRT-PCR. SPC-A1 cells were washed with phosphate-buffered saline (PBS) and lysed with buffer. A biotinylated antisense probe and a control probe were added to the pull-down system before the probes were denatured and hybridized. Non-specifically bound RNA was removed and miRNAs specifically interacting with circRNAs were recovered using Trizol reagent. After reverse transcription, qRT-PCR was performed to testify binding strength.