Protein g sepharose 4 beads

Cells cultured in 6-well plates were harvested and solubilized in lysis buffer (Pierce IP lysis buffer; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After centrifugation, the supernatant was incubated for 12 h at 4 °C with 2 mg of anti-CDK5 (#12134; Cell Signaling Technology, Tokyo, Japan). Following the addition of 30 mL Protein G Sepharose™ 4 beads (GE Healthcare, Chicago, IL, USA), the mixture was incubated for 2 h at 4 °C with rotation. Immune complexes were washed three times with lysis buffer, and the Sepharose beads were boiled for 10 min in sample buffer. Immunoprecipitates were run on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) using 4–20% gradient precast gels (Bio-Rad), followed by western blot analysis with primary antibodies against p35/p25 (#2680, Cell Signaling Technology) and CDK5.

The ABE assay is adapted from Wan and colleagues, with modifications (Wan et al, 2007). Cells were lysed in lysis buffer (LB pH7.4) containing 50 mM Tris–HCl pH7.4, 150 mM NaCl, 1% NP‐40, 1 mM EDTA, and protease inhibitors. For this protocol, all centrifugation steps are carried out at 850 g for 5 min. EGFR was first immunoprecipitated from 100 μg of protein using 5 μg of anti‐EGFR antibody, and complexes of EGFR and anti‐EGFR antibody were bound to Protein G Sepharose 4 beads (#17‐0618‐02, GE Healthcare). 50 mM of N‐ethylmaleimide (NEM, E3876, Sigma) in LB pH7.4 was then added to the immunoprecipitated EGFR and incubated 3 h at 4°C with gentle rotation to block free thiols of cysteine residues. After three washes with LB pH7.4, EGFR was treated with and without (mock as control) 1 M of hydroxylamine (HAM, #379921, Sigma) in LB pH7.4 for 2 h at room temperature with gentle rotation. EGFR was then rinsed three times with LB pH6.2 followed by treatment with 5 μM of BMCC‐Biotin (#21900, Thermo Fischer Scientific) in LB pH6.2 for 2 h at room temperature with gentle rotation. This was followed by three rinses with LB pH7.4 to remove excess biotin, and EGFR was then eluted with reducing sample buffer. Samples were analyzed by SDS–PAGE gels.