HEK293T cells expressing myc-tagged LacZ, CCP5, CCP6, or CCP5 truncated variants were collected after wash with cold D-PBS (Solarbio, D1040, Beijing, China) and then lysed on ice with buffer-1 (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, pH7.4), supplemented with protease inhibitors (Meilun, Shanghai, China). The lysates were centrifuged at 13,000 × g for 10 min at 4 °C, and the supernatants were incubated with the Pierce Anti-c-Myc Magnetic Beads (Thermo Fisher Scientific, #88,844, Massachusetts, USA) at 4 °C overnight. After 3 times wash with buffer-2 (25 mM Tris, 150 mM NaCl, 0.05% Tween-20; pH7.5) and an additional wash with ultrapure water, the beads were boiled in Lane Marker Sample Buffer (Thermo Fisher Scientific, Massachusetts, USA) at 95 °C for 10 min to elute the binding proteins.
To immunoprecipitate CP110 interacting proteins, CP110 antibody was incubated with BeaverBeads™ Protein A/G Magnetic beads (Beaver, Guangzhou, China) with a rotation at a rate of 15 rpm for 1 h at RT. After extensive washing with PBS, the beads were incubated with cell lysates at 4 °C for at least 2 h and then washed with buffer-2 as described above. The beads were then boiled in SDS-PAGE loading buffer at 95 °C for 5 min. The eluted proteins were detected by immunoblotting as described above.
To immunoprecipitate CP110 interacting proteins, CP110 antibody was incubated with BeaverBeads™ Protein A/G Magnetic beads (Beaver, Guangzhou, China) with a rotation at a rate of 15 rpm for 1 h at RT. After extensive washing with PBS, the beads were incubated with cell lysates at 4 °C for at least 2 h and then washed with buffer-2 as described above. The beads were then boiled in SDS-PAGE loading buffer at 95 °C for 5 min. The eluted proteins were detected by immunoblotting as described above.