Plk1 pt210

The cells grown on coverslips were fixed with 3% paraformaldehyde solution at room temperature for 10 min and then permeabilized with 0.5% Triton X-100 at room temperature for 5 min. The cells were incubated with antibody against Aurora B (Santa Cruz Biotechnology; sc-25426), Aurora B-pT232 (Santa Cruz Biotechnology; sc-293127), PLK1 (Santa Cruz Biotechnology; sc-55504), PLK1-pT210 (Abcam; ab39068), Mad2 (Pierce; PA5-21594), BubR1 (BD Biosciences; 612503), histone H3-pS10 (Sigma; 06570), CCAR2 (Bethyl Laboratories; A300-434A), CREST (ImmunoVision, Springdale; HCT-0100), Pericentrin (Abcam; ab28144) or α-Tubulin (Invitrogen; PA5-29444, Sigma; T5168) at 37 °C for 20 min and then incubated with corresponding secondary antibody at 37 °C for 20 min. The nuclei were counterstained with Hoechst 33342 (Invitrogen; H21492). After a final wash with PBS, coverslips were mounted with antifade solution containing para-phenylenediamine and glycerol in PBS.

Cells were lysed on ice for 10 min using NETN lysis buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, 0.5% Nonidet P-40, 50 mM β-glycerophosphate, 10 mM NaF, and 1 mM Na₃VO₄) containing a protease inhibitor cocktail (Millipore; 535140). After centrifugation at 12,000 × g for 5 min, the supernatant was saved as a crude cell extract. This was boiled in Laemmli buffer and loaded onto SDS-polyacrylamide gel. Western blotting was performed according to a standard protocol. The following antibodies were used for western blotting: GAPDH (Santa Cruz Biotechnology; sc-25778), β-actin (Cell Signaling Technology, 4970), FLAG (Sigma; 3165), Myc (Cell Signaling Technology; 2278), Cyclin B1 (Santa Cruz Biotechnology; sc-752), Cyclin D1 (Santa Cruz Biotechnology; sc-8396), Aurora B (Cell Signaling Technology, 3094), Aurora B-pT232 (Santa Cruz Biotechnology; sc-293127), PLK1 (Santa Cruz Biotechnology; sc-55504), PLK1-pT210 (Abcam, ab39068), BubR1 (BD Biosciences, 612503). BubR1-pS670 antibody was provided by C.W. Lee (Sungkyunkwan University). CCAR2 antibody was obtained from immunized rabbit with GST-fused CCAR2 recombinant protein [65 , 66 (link)].

Cells were lysed using lysis buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.5% NP‐40 and cocktail protease inhibitors). The cell lysate was incubated with indicated antibodies at 4°C overnight, followed by the addition of protein G‐coated agarose beads (Invitrogen). After incubation at 4°C for 4 h, beads were washed three times with the lysis buffer. The precipitated components were eluted by boiling the beads in Sodium dodecyl sulfate loading buffer. Western blotting was performed as previously described. Primary antibodies were listed as: METTL16 (Sigama, HPA020352), METTL16 (abconal, A15894), Soga1 (Santa, sc‐514,885), GAPDH (abconal, AC001), anti‐p‐Ser/Thr (BD Biosciences, 612,549), PLK1 (abcam, ab17057), PLK1‐pT210 (abcam, ab155095), Flag (CST, 14793), Flag (Sigma, F3165), HA (abconal, AE008), HA (abconal, AE036).