Ab9344

Antibodies used in the study were as follows: anti-Flag (F3165/F7425, Sigma), anti-Myc (9B11/71D10, Cell Signaling Technology (CST)), anti-HA (16B12, Biolegend and H6908, Sigma), anti-TRAF2 (sc-136999, Santa Cruz), anti-Rab5 (sc-46692, Santa Cruz), anti-Rab7 (sc-376362, Santa Cruz), anti-Sprouty 2 (sc-100862, Santa Cruz/ab85670, Abcam), anti-EGFR (sc-373746, Santa Cruz), anti-p-Tyr (9411, CST), anti-p-Ser/Thr (ab9344, Abcam and 05-368, Sigma), anti-Ser (sc-81514, Santa Cruz), anti-Thr (sc-5267, Santa Cruz), anti-Ubiquitin (sc-8017, Santa Cruz), anti-β-Actin (AC026, Abclonal), anti-GFP (AE011, Abclonal), anti-glutathione S-transferase (GST) (2622 S, CST), anti-V5 (R960-25, Thermo Fisher and 30801ES10, Yeasen), anti-Mouse/Rabbit IgG-peroxidase secondary antibody (A0545/A9044, Sigma), anti-Mouse IgG (light chain specific)-peroxidase secondary antibody (115-005-174, Jackson), and anti-DYRK1A polyclonal antibody has been previously described [30 (link)].

Cells were lysed in ice cold buffer (150 mM NaCl, 1.5 mM MgCl₂, 10 mM KCl, 20 mM Tris [pH 7.9], 0.5 mM EDTA, 10% glycerol, 1 mM DTT, 0.1% PMSF, EDTA-free complete protease inhibitor mixture (Roche) and 0.5% Nonidet P-40) for 15 min. Total cell lysates were cleared by centrifugation at 17,000×g/4 °C for 10 min. Supernatant was mixed with indicated antibodies, and rotated for 2 h at 4 °C. Beads were washed by the same buffer for three times (100 beads volume each time). Immunoprecipitates were eluted by 2X SDS-PAGE sample buffer for subsequent analyses. For CDK2 kinase assay, endogenous CDK2 protein was pulled down by anti-CDK2 antibody immobilized on protein A/G agarose beads. Recombinant human histone H1 (Abcam, ab198676) was used as substrate (20 mM HEPES/KOH pH 7.9, 5 mM MgCl₂, 1 mM DTT and 1 mM ATP). Reactions were terminated by 2X SDS-PAGE sample buffer, and phosphorylated H1 protein was detected by anti-phospho-T/S-Pro (Abcam, ab9344).