Human ago2 antibody or control igg

RIP assay was conducted utilizing the EZ-Magna RIP kit (Millipore, Billerica, MA) according to the manufacturer’s protocol. A549 and SPCA1 cells at 80–90% confluence were obtained, and then lysed in complete RIP lysis buffer. Cell extract was processed at 4 °C for about 6 h with RIP buffer which contained human Ago2 antibody or control IgG (Millipore) coated magnetic beads. After beads washed, the RNA complexes were cultured with Proteinase K to digest proteins. RNA concentration was measured though employing a NanoDrop spectrophotometer (Thermo Scientific), with the quality assessed by a bioanalyser (Agilent, Santa Clara, CA). Finally, the immunoprecipitated RNAs were purified and analysed by RT-qPCR.

A Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) was used based on provided directions in order to assess interactions between miR-302b and lncZEB1-AS1. Briefly, cellular lysates were mixed at 4 °C overnight with magnetic beads coated with a human AGO2 antibody or control IgG (Millipore). These beads were then collected, and qRT-PCR was used in order to analyze RNA enrichment in the precipitated fraction.