Co ip buffer

Cultured cells (10⁷) were lysed in 500 μl co-IP Buffer (Cell lysis buffer for western and IP, Beyotime, China), supplemented with protease inhibitor cocktail Tablets (Roche, Switzerland), Pierce^TM phosphatase inhibitor (Thermo Scientific), and Protector RNase Inhibitor (Roche). Next, the cell lysates were incubated with 800 pmol of biotinylated DNA probes for circ_CEA (5′-GCCCATCAGTCTTCCTGAAA-3′) or scramble probes (5′-ATCTAATAGCTCCACGTGCC-3′) at 4 °C overnight. Next, Streptavidin C1 magnetic beads (Invitrogen) were blocked with 2 mg/mL BSA at room temperature for 1 hr, and then added to each binding reaction and incubated at room temperature for 1 hr. After washing in co-IP Buffer, beads were incubated with Non-Reducing Lane Marker Sample Buffer (Thermo Scientific) at room temperature for 10 min to elute the bound proteins. The proteins were detected by western blotting with the antibodies against p-p53 ser315, p53, CDK1, and FoxO3 (Cell Signaling Technology).

DEFs were cotransfected with Myc-duRIG-I, Flag-duTRIM35, or an empty vector in the presence or absence of HA-Ub or its mutations. After transfection, cells were harvested with Co-IP buffer (Beyotime), and cell lysates were immunoprecipitated with anti-Myc MAb, followed by incubating with protein A/G plus-agarose (Santa Cruz Biotechnology). After washing with lysis buffer, the immunoprecipitates were analyzed by standard immunoblot procedures with the appropriate antibodies.

After treatment, the cells were scraped and incubated with Co-IP buffer (Beyotime Institute of Biotechnology, Jiangsu, China) adding a commercial protease inhibitor cocktail (Sigma-Aldrich) at 4 °C for 20 min. Then, the lyses were centrifuged at 16,000 rpm; 4 °C for 20 min. Primary antibody (1:100 dilutions) was added into the supernatants (400 μL) and incubated at 4 °C with gentle rocking overnight. Immunocomplexes were incubated with 40 μL protein A-agarose beads (fast flow) with gentle rocking for 6 h at 4 °C, and resuspended in 50 μL SDS sample buffer (2×) followed by boiling for 5 min. Finally, the samples were separated by SDS-PAGE and analyzed using the Western blot assay.