Mouse igg1 isotype control

Pierce^TM Classic Magnetic IP/Co-IP kit (Thermo Scientific) was used for RIP assay. Cultured cells (10⁷) were lysed in 800 μl Pierce IP Lysis/Wash Buffer supplemented with protease inhibitor cocktail Tablets (Roche), and Protector RNase Inhibitor (Roche). The cell lysate was incubated with antibodies against p53 (#9282), CDK1, or FoxO3 (Cell Signaling Technology) at 4 °C overnight. Mouse IgG1 Isotype control and rabbit mAb IgG XP Isotype control (Cell Signaling Technology) were used as a negative control. Next, 0.25 mg of Pierce Protein A/G Magnetic Beads were added to each sample and incubated at room temperature for 1 hr. Then the pellets were collected, washed with Pierce IP Lysis/Wash Buffer, and then resuspended in TRIzol Reagent (Invitrogen). RNAs were isolated from the pellets and circ_CEA enrichment was evaluated by qRT-PCR assay.

NRCMs or heart tissues were lysed with IGEPAL CA-630 buffer (50 mM Tris-HCl, pH 7.4, [Sigma-Aldrich, T5030], 1% IGEPAL CA-630 [Sigma-Aldrich, I8896], 10 mM EDTA, 150 mM NaCl, 50 mM NaF, 1 μM leupeptin [Sigma-Aldrich, L5793], 0.1 μM aprotinin [Sigma-Aldrich, SRE0050]). Samples were incubated for 2 h with primary antibody after being precleared with 30 μL PureProteome™ Protein A/G Mix Magnetic Beads (Merck Millipore, LSKMAGAG10) at 4 °C. Cell lysates were also subjected to immunoprecipitation with either mouse IgG1 isotype control (Cell Signaling Technology, 5415) or rabbit IgG isotype control (Cell Signaling Technology, 3900) according to the immunoglobulin type of primary antibody. After immunoprecipitation, the samples were washed five times with TBS. They were then eluted with glycine-HCl (0.1 M, pH 3.5) and the immunoprecipitates were subjected to immunoblotting using specific primary antibodies.

Co-IP assay was conducted with Pierce Classic Magnetic IP/Co-IP Kit (Thermo Scientific). Cultured cells (10⁷) were lysed in 800 μl Pierce IP Lysis/Wash Buffer supplemented with protease inhibitor cocktail Tablets (Roche). The cell lysate was incubated with the anti-CDK1 antibody (Cell Signaling Technology) at 4 °C overnight. Mouse IgG1 Isotype control (Cell Signaling Technology) was used as a negative control. Next, 0.25 mg of Pierce Protein A/G Magnetic Beads were added to each sample and incubated at room temperature for 1 hr. Then the pellets were collected, washed with Pierce IP Lysis/Wash Buffer, and then incubated with Non-Reducing Lane Marker Sample Buffer (Thermo Scientific) at room temperature for 10 min to elute the bound proteins. The p53 proteins co-precipitated with anti-CDK1 antibody were detected by western blotting. To avoid the detection of IgG heavy and light chains, VeriBlot for IP detection reagents (Abcam) were used as secondary antibodies.