Immunoprecipitation kit

The RIP assay was carried out using an Immunoprecipitation Kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. AGS cells were harvested and lysed in RIP lysis buffer with a ribonuclease inhibitor and protease inhibitor cocktail. Then, magnetic beads were configured for immunoprecipitation, and protein antibodies SMAD2/SP1/IgG were added for a further 30 min incubation at room temperature. Then, anti-SMAD2/SP1 or anti-IgG magnetic bead antibodies were added to the supernatant of the AGS cell lysate after centrifugation, which were incubated overnight at 4 °C. After the beads were washed with wash buffer, immunocomplexes of SMAD2/SP1/IgG and RNAs were de-crosslinked with protease K buffer at 55 °C for 30 min. The immunoprecipitated RNAs were then purified using TRIzol and ethanol precipitation and subjected to qRT-PCR analysis.

RIP assay was performed using the Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Cell extracts of SW480 and HT-29 cells were incubated with protein A/G sepharose beads at 4 °C. Immunoprecipitated RNAs were subjected to real-time PCR.

RIP and IP were performed to confirm the RNA–protein and protein–protein interactions using RIP Kit (17-700, Millipore) and Immunoprecipitation Kit (ab206996) according to the manufacturer’s guidance. RIP related details had been described in our previous studies^{33 (link),34} . The IP assay consists of four steps, including antibody binding, beads preparation, bead capture, and elution. The volume of the antibody binding system was made up to 500 µl with lysis buffer containing the protease inhibitor cocktail and gently mixed for 4 h; the protein A/G sepharose (30 µl/reaction) was washed twice with wash buffer, centrifuged at 2000 × g for 2 min and aspirated the supernatant between washes.