Lambda phosphatase

Cells were cultured in medium containing 100 ng/ml nocodazole (Sigma, M1404) to arrest in mitosis for 16 h. Mitotic cells were isolated by mechanical shake-off. Then, the cell lysates were reacted with lambda phosphatase (NEB, P0753S) in lambda phosphatase buffer (20 mM Tris-HCl, pH 7.6; 250 mM NaCl, and 0.5% NP-40) supplemented with 2 mM MnCl₂ at room temperature for 2 h; the reaction was stopped with Laemmli sample buffer.

D.Mel-2 cells were co-transfected with pAct5-Ana2-FLAG and either pAct5-Plk4-NDKD (kinase-dead) or pAct5-Plk4-ND (active) in a 12-well plate. Approximately 1 × 10⁶ cells were collected from each well 24 h post-transfection and briefly rinsed in PBS. Cells were then lysed in RIPA buffer (50 mM Tris–HCl pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% Na deoxycholate; 0.1% SDS; 1× Complete EDTA-free protease inhibitor cocktail tablets from Roche) on ice for 15 min. Lysates were cleared by centrifugation and supplemented with 1× Lambda-phosphatase buffer and 1× MnCl₂ solution provided along with Lambda phosphatase (New England Biolabs, catalogue number P0753S). Two samples (one of Ana2-FLAG + Plk4-NDKD and one of Ana2-FLAG + Plk4-ND) were mock-treated (no phosphatase added), while one sample (Ana2-FLAG + Plk4-ND) was treated with 200 U (0.5 µl) Lambda phosphatase for 30 min at 30°C. All samples were then boiled in Laemmli sample buffer and analysed by immunoblotting.