Non reducing lane marker sample 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).

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.

PD-1 IP studies were performed using Pierce Protein A/G Plus agarose beads (Invitrogen), according to the manufacturer’s instructions. Briefly, cells were lysed in ice-cold buffer (150 mM NaCl, 50 mM Tris-HCI and protease inhibitor cocktail, Roche), sonicated (three 10 s bursts), vortexed for 2 h at 4 °C in 2% Nonidet P-40 (NP-40, Sigma), and centrifuged. B16-F10 lysates were concentrated using Microcon-10 Ultracel PL-10 filter columns, as above. Cell lysates were precleared for 2 h at 4 °C by incubation with Protein A/G Plus agarose beads previously blocked in ice-cold buffer supplemented with 1% BSA for 1 h at 4 °C, incubated with anti-mouse PD-1 (4–6 µg, RMP1-14)^{53 (link)} or rat IgG2a isotype control abs for 2 h at 4 °C, and then with Protein A/G Plus agarose beads overnight at 4 °C under continuous rotation. Supernatants were kept for assessment of IP efficiency, Protein A/G Plus agarose beads washed extensively, and IP products eluted in ice-cold buffer, as above, supplemented with 1.5 × Non-reducing Lane Marker Sample Buffer (Thermo Fisher), and boiled at 100 °C for 7min^{54 (link)}. IP products were then analyzed by immunoblotting, as above, or resolved in 7.5% SDS-PAGE gels, stained with Colloidal Coomassie Blue (Bio-Rad), and subjected to MS sequencing, as described below.