Arabidopsis seedlings were grounded in the presence of liquid nitrogen to fine powder and extracted with buffer containing 50 mM HEPES (NaOH, pH 7.5), 150 mM NaCl, 50 mM β-glycerophosphate, 2 mM DTT, 1% Triton X-100 and 10% glycerol, with EDTA-free protease inhibitors (Roche). After centrifugation for 10 min at 20,000 g, the supernatant was isolated and used as protein samples for Phostag gel analysis. For dephosphorylation of CBL1/9 and CBL2/3, 50 μL supernatant was incubated with 1 μL λ-PPase (NEB) and 5 μL 10 mM MnCl2 under 30 °C for the indicated times. For dephosphorylation of CIPK9-3Flag, CIPK23-3Flag, AKT1-3Flag proteins, the supernatant was incubated with 10 μL prewashed anti-Flag M2 agarose beads (Sigma-Aldrich) for 1 h at 4 °C on a roller shaker. The beads were then washed three times with the extraction buffer described above. The protein bound beads were incubated with 1 μL λ-PPase (NEB) and 5 μL 10 mM MnCl2 under 30 °C for 30 min. The dephosphorylation reaction was stopped by adding 2× SDS loading buffer and boiled for 10 min.
λ ppase
Manufactured by New England Biolabs
Sourced in United Kingdom, United States
λ-PPase is a highly specific and active serine/threonine protein phosphatase. It catalyzes the removal of phosphate groups from phosphorylated proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (2 mentions)
Nickel nitriloacetic acid, by Qiagen (2 mentions)
Sf9 insect cells, by American Type Culture Collection (2 mentions)
Phos tag acrylamide, by Fujifilm (2 mentions)
Edta free protease inhibitor, by Merck Group (2 mentions)
Anti flag m2 agarose bead, by Merck Group (1 mentions)
Okadaic acid, by Merck Group (1 mentions)
Sb203580, by Enzo Life Sciences (1 mentions)
5z 7 oxozeaenol, by Bio-Techne (1 mentions)
Rapamycin, by Enzo Life Sciences (1 mentions)
Okadaic acid, by Enzo Life Sciences (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Ebio7979, by Thermo Fisher Scientific (1 mentions)
Sp600125, by Enzo Life Sciences (1 mentions)
U0126, by Enzo Life Sciences (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Mg132, by Merck Group (1 mentions)
Thymidine, by Merck Group (1 mentions)
23 protocols using λ ppase
1
Phospho-protein Dephosphorylation Protocol
2
Dephosphorylation of Recombinant IRE1α
3
Dephosphorylation of Recombinant IRE1α
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A construct containing the cytosolic kinase and RNase domains of human IRE1α (residues 547–977, IRE1α*) was expressed in SF9 insect cells (ATCC, #CRL-1711) by using the Bac-to-Bac baculovirus expression system (Invitrogen) with a His6 tag at the N-terminus and purified with a nickel–nitriloacetic acid (Qiagen) column. To generate dP-IRE1α*, we removed basal phosphorylation sites by incubating IRE1α* with λ-PPase (New England Biolabs) at a molar ratio of 5:1 (IRE1α*: λ-PPase) in 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MnCl2, 2 mM dithiothreitol (DTT) and 0.01% Brij 35 detergent (v/v) overnight at 4°C. Dephosphorylation was verified through western blotting using a universal phospho-protein detection agent (biotin-pIMAGO (Tymora Analytical Operations, Cat. No.: FL800) and imaged using a streptavidin-linked fluorophore.
4
Phosphorylation Analysis by λ-PPase
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Lambda protein phosphatase (λ-PPase) could remove the phosphate groups (dephosphorylate). In order to verify whether the diffused bands were phosphorylated or not, λ-PPase was used. Granulosa cells were cultured in the medium supplemented with IGF-1 for 24 h. Cells were collected, lysed and divided into two equal parts. One was treated with 1 µL λ-PPase (400U/µl, New England Biolabs, Beverly, MA, United Kingdom) at 30 °C for 2 h and the other was subjected to the same treatment, except for λ-PPase. After adding 10 µL 5× SDS sample buffer to two samples, samples were boiled for 10 min.
5
Phosphatase Activity Assay with Phos-tag Gel
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Cells were collected from a 35-mm dish and rinsed twice with ice-cold HBS consisting of 20 mM Hepes, pH 7.4, and 150 mM NaCl. They were subsequently lysed on ice for 15 min with RIPA buffer, containing 1% NP-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, and complete EDTA-free protease inhibitors (Roche). Lysates were centrifuged at 20,000 g for 15 min, and supernatants were supplemented with 10× PMP buffer, 10× MnCl2, and 400 U λ-PPase (New England Biolabs, Inc.). The reactions were kept at 30°C for 30 min before being quenched with SDS sample buffer. Phos-tag gel electrophoresis was performed according to the manufacturer’s protocol (Wako Pure Chemical Industries). In brief, 8% separating gel was prepared with 25 µM Phos-tag Acrylamide AAL-107 and 50 µM MnCl2. We then mixed thoroughly, allowed the gel to polymerize for 1 h, and prepared the normal stacking gel. Samples were run at low voltage around 60 V until the Bromophenol blue reached the bottom. The gel was then soaked in transfer buffer containing 1 mmol/liter EDTA for 10 min and transfer buffer without EDTA for another 10 min before transferring to PVDF membrane for antibody blotting.
6
Phosphatase and Kinase Assays
7
Phosphorylation of PRC2 complex by LATS kinases
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Recombinant EZH2/EED/SUZ12/RbAp48/AEBP2 complex (BPS Bioscience, San Diego, CA, USA) (700 ng) was incubated with 100 ng of recombinant LATS2 or LATS1 kinases (Carna Biosciences, Hyogo, Japan) at 30°C for 30 min with kinase reaction buffer (5 mM MOPS-NaOH [pH 7.2], 5 mM magnesium chloride, 1 mM EGTA, 0.4 mM EDTA, 5 mM glycerol 2-phosphate, 50 μM DTT, and 50 μM ATP). For protein phosphatase (PPase) assay, 200 U of λ-PPase (New England Biolabs, Ipswich, MA, USA) was added to the kinase reaction tube. Each reaction was carried out in a 25 μl volume. The reaction was stopped by addition of 4× Laemmli sample buffer. Proteins were separated by SDS-PAGE in gels containing 50 μM Phos-tag acrylamide (WAKO, Osaka, Japan) and subjected to western blotting.
8
Investigating Foxp3 Regulation by NLK
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The following antibodies were used: rat anti-Foxp3 clone PCH101 and mouse anti-Foxp3 clone eBio7979 (eBioscience), mouse anti-Flag (Sigma-Aldrich), mouse anti-hemaglutinin (HA) clone 12CAS, rabbit anti-NLK (H-100) and goat anti-Actin (I-19) (Santa Cruz Biotechnology), and anti-HSP90 was purchased from Professor Ineke Braakman (UMC Utrecht, Utrecht, the Netherlands).
The following reagents were used: Phos-tag™ Acrylamide (WAKO Chemicals GmbH), Okadaic acid,SB203580">SB203580 , SP600125">SP600125 , U-0126 and Rapamycin (Enzo Lifesciences), PKB inhibitor VIII (Calbiochem), 5Z-7-Oxozeaenol and BIO (Tocris Bioscience, Bristol, United Kingdom, λPPAse (New England BioLabs) and recombinant GST-NLK active protein (Sigma-Aldrich).
The following reagents were used: Phos-tag™ Acrylamide (WAKO Chemicals GmbH), Okadaic acid,
9
Cell Signaling Protein Analysis Protocol
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Transfection reagents Lipofectamine 2000 and Lipofectamine 3000 were purchased from Invitrogen, RNA synthesis inhibitor actinomycin D, protease inhibitor MG132, lysosomal inhibitors bafilomycin and hydroxychloroquine, CDK inhibitor RO-3306, and thymidine were purchased from Sigma-Aldrich. λPPase was purchased from New England BioLabs. Anti-CELF6 (ab173282) and anti-GFP (ab1218) were obtained from Abcam, anti-p21Waf1/Cip1 (#2947), anti-p27Kip1(#3686), anti-Gadd45α (#4632), anti-Cyclin B1(#12231), anti-β-TrCP (#4394), anti-Wee1 (#13084), anti-cleaved PARP (#5625), anti-cleaved Casp3 (#9661), anti-cyclin E1 (#20808), and anti-Ubiquitin (#3936) antibodies were purchased from Cell Signaling Technology, anti-P53 (10442-1-AP), anti-GFP (66002-1-1 g), anti-His (66005-1-1 g), anti-β-tubulin (60008-1-1 g), and anti-GAPDH (10494-1-AP) were obtained from Proteintech, anti-LC3B (L7543) was obtained from Sigma-Aldrich.
10
Dephosphorylation of Pex14p by Lambda Phosphatase
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Pex14pTPA affinity-purified from Triton X-100 extracts was mixed with 10x phosphatase buffer (0.5 M HEPES, 1 M NaCl, 20 mM DTT, 0.1% Brij 35, pH 7.5), MnCl2 (1 mM final concentration), and 800 units of lambda protein phosphatase (λ-PPase; New England Biolabs) and incubated for 20 min at 30°C under slight agitation. As control, the reaction was performed without λ-PPase using the same amount of Pex14pTPA. Reactions were stopped by adding SDS sample buffer and proteins were separated by Phos-tag SDS-PAGE using a standard 10% SDS polyacrylamide gel additionally containing 50 μM Phos-tag reagent (Phos-tag acrylamide AAL-107; Wako Pure Chemical Industries, Ltd., Japan) and 100 mM MnCl2 in the separation gel. Electrophoresis was carried out at 80 V for 4 h. Subsequently, the gel was incubated for 20 min in Western blot transfer buffer [25 mM Tris, 192 mM glycine, 20% (v/v) methanol] containing 10 mM EDTA to quench the Phos-tag reagent and washed for 10 min in transfer buffer without EDTA. Proteins were transferred to a polyvinylidene difluoride membrane at a constant voltage of 30 V and 4°C for 14 h.
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