λ protein phosphatase

When calf intestine phosphatase (CIAP) (New England Biolabs: NEB) was used, Xenopus embryos overexpressing HA-tagged Xl_Nemp1 (Xl_Nemp1-HA) were lysed in lysis buffer A. Lysates were incubated with anti-HA antibody at 4 °C for 1 h, then added with protein G-agarose beads, and incubated for another 1.5 h. The beads were washed 3 times with lysis buffer A, once with NEBuffer 3 (NEB), and incubated in NEBuffer 3 containing 0.5 u/ml of CIAP for 3 h at room temperature. When λ protein phosphatase (NEB) was used, Xenopus embryos overexpressing mouse Nemp1-HA (Mm_Nemp1-HA) were lysed in lysis buffer A without EDTA. Lysates were incubated with λ protein phosphatase in NEBuffer for Protein MetalloPhosphatases (NEB) for 45 min at 30 °C. Treated samples were analyzed by western blotting with anti-HA antibody.

After treatment, cells were lysed in RIPA buffer (50 mm Tris pH 7.4, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP‐40, 1 mm sodium vanadate, and 50 mm NaF). Half of the sample was directly heated with 2 × LDS loading buffer (ThermoFisher, Waltham, MA, USA), while the other half was treated with a 1 : 1 mixture of Alkaline phosphatase (ThermoFisher): λ protein phosphatase (New England Biolabs, Ipswich, MA, USA) for 30 min at 30 °C, and then heated with loading buffer. Lysates were then resolved by both SDS/PAGE and Phos‐tag PAGE. Phos‐tag (Wako Pure Chemical Industries, Richmond, VA, USA) analysis was performed according to the manufacturer's protocol using a neutral‐pH gel system and a Zinc(II) complex. The antibodies used in this study are listed in Table S2. Imaging and quantification of western blots were performed with a UVITEC imaging system and the imagej program.

About 10 μmol purified CPC were incubated with 2.5 μmol λ protein phosphatase (New England Biolabs) in PMP buffer (50 mM Hepes, 2 mM DTT, 0.01% Brij 35; New England Biolabs) supplemented to 500 mM NaCl to promote solubility of the CPC and in the presence of 1 mM MnCl₂. Reactions were stopped by addition of Laemmli sample buffer, after which samples were boiled for 10 min and separated by SDS-PAGE.

Native kinetochore particles were purified (see Kinetochore purification) and kept bound to Dynabeads (Invitrogen). Beads were washed once with phosphatase buffer (buffer H, 1 mM MnCl), and then resuspended in phosphatase buffer with 200 U λ protein phosphatase (New England Biolabs) at 30°C for 20 min. Control samples contained phosphatase inhibitors. Kinetochores were eluted by boiling beads in sample buffer containing SDS, and phosphorylation was analyzed via immunoblotting.

Phosphatase treatment was performed according to Suttangkakul et al. (2011) (link) with minor modification. Seven-day-old YFP-ATG1a and YFP-ATG1a/KIN10-OE seedling were homogenized in ice-cold protein extraction buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche). Samples were placed on ice for 30 min, and then centrifuged for 30 min at 11,000 g. The supernatant was incubated with λ protein phosphatase (New England Biolabs) with or without addition of phosphatase inhibitor PhosSTOP (Roche) for 30 min at 30°C. 2 × SDS-PAGE sample buffer was added to the total sample and heated to 95°C for 5 min.