The 7.2 kb Csf1r reporter construct previously used to generate the Csf1r-EGFP mice (26 (link)) was digested with ApaI and SalI (NEB) to remove EGFP before gel purification using the QIAquick gel extraction kit (Qiagen). Overhangs were removed with Mungbean nuclease (NEB) and DNA was purified using QIAGEN MinElute columns (Qiagen), then dephosphorylated using thermosensitive alkaline phosphatase (Promega). A construct encoding the fluorescent protein Csf1r-mApple (34 (link)) was digested with SmaI and AflII, similarly purified, and overhangs removed before both constructs were precipitated with EtOH/NaOAc and then ligated with T4 ligase (NEB) at 16°C overnight. The resulting Csf1r-mApple construct was transformed into DH5α competent cells. The Csf1r-rtTA-M2 construct utilizing the same 7.2 kb mouse Csf1r promoter region was used previously to generate Csf1r-rtTA transgenic mice (35 (link)) For generation of transgenic mice, plasmid backbones were removed by digestion with DrdI/PvuI (Csf1r-mApple, NEB) and SalI/MluI (Csf1r-rtTA, Promega/NEB) and then gel-purified using a QIAquick gel extraction kit. DNA was then further purified using AMPure XP beads (Agencourt) according to the instructions.
Thermosensitive alkaline phosphatase
Manufactured by Promega
Thermosensitive alkaline phosphatase is an enzyme designed for use in molecular biology applications. It catalyzes the removal of phosphate groups from various molecules, including nucleic acids and proteins. The enzyme is thermosensitive, meaning its activity is temperature-dependent.
Automatically generated - may contain errors
Lab products found in correlation
Mung bean nuclease, by New England Biolabs (1 mentions)
Minelute column, by Qiagen (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
T7 rna polymerase, by Takara Bio (1 mentions)
Rnase inhibitor, by Takara Bio (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
Quantum prep pcr kleen spin columns, by Bio-Rad (1 mentions)
Pureyield plasmid miniprep, by Promega (1 mentions)
Modified trypsin, by Promega (1 mentions)
Cdk4 cyclin d1, by Thermo Fisher Scientific (1 mentions)
Cdk6 cyclin d1, by Thermo Fisher Scientific (1 mentions)
Cdk9 cyclink, by Thermo Fisher Scientific (1 mentions)
Gblocks gene fragment, by Integrated DNA Technologies (1 mentions)
Gibson assembly mix, by New England Biolabs (1 mentions)
Dna clean and concentrator kit, by Zymo Research (1 mentions)
6 protocols using thermosensitive alkaline phosphatase
1
Generation of Csf1r-mApple reporter mice
2
Identification of LATS1 Phosphorylation Motifs
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Thirty minutes after release from nocodazole-induced prometaphase arrest, HeLa cells were collected and immunoprecipitated with an anti-CDC27 antibody. In vitro kinase reactions were then performed using the immunoprecipitates and cold ATP. The resulting samples were analyzed by nano-liquid chromatography/tandem mass spectrometry, as described previously [10 (link)]. The LATS1 phosphorylation motifs were identified using in vitro kinase assays, as described previously [38 (link)]. Briefly, HeLa cell lysates were dephosphorylated with thermo-sensitive alkaline phosphatase (Promega). After heat inactivation of the phosphatase, the lysates were diluted with 40 mM Tris-HCl (pH 7.5) containing 20 mM MgCl2 and 1 mM ATP, and then reacted with recombinant LATS1 for 3 h at 37°C. The reaction mixture was denatured with 8 M urea followed by reductive alkylation and tryptic digestion. The phosphopeptides were enriched with hydroxyl acid-modified metal oxide chromatography [18 (link)] using titania and analyzed via nano-liquid chromatography/tandem mass spectrometry. Based on the LATS1 substrate sites identified in vitro, phosphorylation motifs were obtained using motif-x [39 (link)].
3
Preparation of Precursor tRNAs from Physcomitrella
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DNAs representing precursors of chloroplast tRNAPhe, mitochondrial tRNACys, and nuclear tRNAAsp were amplified by PCR from P. patens genomic DNA with oligonucleotide pairs T7-cpF5 and cpF3, T7-mtC5 and mtC3, and T7-nuDGTC5 and nuDGTC, respectively (Table S2 ). The 5′ oligonucleotides contained the T7 promoter sequence. The pre-tRNAPhe, pre-tRNACys, and nuclear pre-tRNAAsp were respectively 142, 153, and 140 nt long and were designed with respective 5′ leaders of 42, 51, and 40 nt and 3′ trailers of 27, 32, and 25 nt. Amplified DNAs (200 ng) were transcribed with T7 RNA polymerase (TaKaRa) for 1 h at 37°C, then digested with 5 U of RNase-free DNase I (TaKaRa) and 20 U of RNase Inhibitor (TaKaRa) for 15 min at 37°C. Synthesized pre-tRNAs were purified by phenol chloroform extraction.
For preparation of chloroplast pre-tRNAArg isoacceptors, the corresponding genes were cloned and in vitro transcribed. The obtained pre-tRNAs were dephosphorylated with Thermo Sensitive Alkaline Phosphatase (Promega) and were 5′-end radiolabeled with [γ32P] ATP and polynucleotidekinase (TaKaRa). Labeled RNAs were purified from the gel after 8% polyacrylamide gel electrophoresis (PAGE) containing 7M urea.
For preparation of chloroplast pre-tRNAArg isoacceptors, the corresponding genes were cloned and in vitro transcribed. The obtained pre-tRNAs were dephosphorylated with Thermo Sensitive Alkaline Phosphatase (Promega) and were 5′-end radiolabeled with [γ32P] ATP and polynucleotidekinase (TaKaRa). Labeled RNAs were purified from the gel after 8% polyacrylamide gel electrophoresis (PAGE) containing 7M urea.
4
Plasmid Preparation and Enzymatic Manipulation
5
Quantifying Kinase Activity Using Isotope-Labeled Formaldehyde
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Titanium dioxide (titania) particle (10 μm diameter) were purchased from GL Sciences (Tokyo, Japan). Thermo-sensitive alkaline phosphatase (TSAP) and modified trypsin were obtained from Promega (Madison, WI). [2H2, 13C]Formaldehyde was purchased from ISOTEC (Miamisburg, OH). Recombinant human protein kinases were from Carna Biosciences (Kobe, Japan), with the exception of KHS1, SGK496, NIK, CDK6/Cyclin D1, CDK9/Cyclin K, ALK1, CDK4/Cyclin D1, BARK1 and MPSK1 (Invitrogen, Carlsbad, CA) and GPRK5, FRAP, DAPK2, PKG1a, STK33, TAO1 and ULT3 (Millipore, Billerica, MA). More details on kinases are shown in Supplementary Table S5 and Supplementary Information. All other reagents were from WAKO Chemicals (Osaka, Japan).
6
Codon-Optimized Heterologous Gene Expression in L. lactis
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Genes were codon-optimized using Integrated DNA Technologies’ codon optimization tool for L. lactis cremoris. The codon-optimized genes were amplified from synthesized Gblocks® gene fragments (Integrated DNA Technologies) using the KOD-Xtreme kit (Merck). The PCR products were DpnI-treated for at least 2 h and then cleaned up and concentrated using the DNA clean and concentrator kit (Zymo research). pNZ8148 were digested with restriction enzymes for at least 5 h at 37 °C. They were then incubated with thermosensitive alkaline phosphatase TSAP (Promega) for 2 h before they were cleaned and concentrated. The genes were then assembled into the vectors using Gibson assembly mix (New England Biolabs) for 1 h at 50 °C. 2 μL of the Gibson assembly mixture was added into 50 μL of electrocompetent NZ9000 cells and electroporated using 0.1 cm cuvette at 1800 V. 1 mL of GM17 media with 20 mM MgCl2 and 2 mM CaCl2 was added immediately after electroporation. The cuvette was kept on ice for 5 min before incubating the cells at 30 °C for 1–2 h. The cells were centrifuged and resuspended in 100 μL of media before they were plated out on M17 with 0.5% glucose (GM17) agar with 10 µg/mL chloramphenicol and incubated at 30 °C for 2 days. Colonies were screened for the correct construct before isolation and sequencing of the plasmids were performed.
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