Protein metallophosphatase buffer

For immunofluorescence, DIV10 rat hippocampal neurons were fixed for 10 minutes in 1% PFA, washed thoroughly in PBS followed by 0.9% saline, then treated for 3 hours with phosphatase buffer +/− enzyme at 37°C. Calf intestinal phosphatase (CIP, New England Biolabs) was used at a concentration of 1 unit/μl in 1x NEB Buffer 3 (New England Biolabs). Lambda phosphatase was used at a concentration of 40 units/μl in 1x Protein MetalloPhosphatase Buffer with 1 mM MnCl₂ (New England Biolabs).
For Western blots, neurons were lysed in 2% SDS/25 mM Tris as described above then passed three times through a Microcon YM-10 ultracentrifuge filter (Amicon) to remove SDS. Each centrifugation step was done at 12,000 rpm for 45–60 minutes at 4°C followed by reconstitution of the filtrate in 10x volume of fresh 25 mM Tris. Washed lysate was treated with phosphatase buffer +/− enzyme for one hour in a 40 μl reaction. For CIP, 25 μg lysate was incubated with 30 units of enzyme in NEB Buffer 3 at 37°C. For Lambda phosphatase, 25 μg lysate was incubated with 400 units of enzyme in PMP buffer with 1 mM MnCl₂ at 30°C. Samples were subsequently loaded onto SDS-PAGE gels and blotted as described above.

Cells treated with the drugs indicated in the Figure legends were collected, washed once with PBS then lysed in Lysis Buffer without phosphatase inhibitors (50 mM HEPES, 1 mM EGTA, 1 mM MgCl₂, 100 mM KCl, 10% glycerol, 0.05% NP-40, 1 mM dithiothreitol, 1X Complete EDTA-free protease inhibitor cocktail (Roche), 1 mM phenylmethylsulfonyl fluoride, pH 7.4) for 10 min on ice. Cellular debris was removed by centrifugation and protein concentrations were measured by Bradford assay (Bio-Rad) and normalized. Cell lysates were supplemented with 1X Protein MetalloPhosphatase buffer (New England Biolabs) and 1 mM MnCl₂. 1 μL of Lambda Protein Phosphatase (New England Biolabs) or 1 μL of phosphatase inhibitor mix (20 mM β-glycerophosphate, 1 mM sodium fluoride, and 0.4 mM sodium orthovanadate) was added to each 50 μL reaction. After incubation at 30°C for 30 min, reactions were stopped by addition of 2X Laemmli buffer. Samples were analyzed by SDS-PAGE and Western blot.

HeLa cells were treated with 330 nM nocodazole overnight and mitotic cells were harvested by shake-off. Cells were washed with PBS and then lysed on ice for 45 min in HEPES/Triton X-100 lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM MgCl₂, 1X Complete EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF). Cellular debris was removed by centrifugation and the resulting lysate was split into three parts. One sample was left untreated and the other two were supplemented with 1X Protein MetalloPhosphatase buffer (New England Biolabs) and 1 mM MnCl₂ only or together with 1 μl Lambda Protein Phosphatase (New England Biolabs). After incubation at 30°C for 30 min, reactions were stopped by addition of 2X Laemmli sample buffer and heated at 95°C for 5 min. Samples were analyzed by SDS-PAGE and Western blot.