m0290  (New England Biolabs)

Journal: Genes & Development

Article Title: DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

doi: 10.1101/gad.282616.116

Figure Lengend Snippet: DUSP11 directly dephosphorylates the BLV pre-miRNAs and 5p miRNAs. ( A ) Immunoblot analysis to confirm expression of DUSP11 and DUSP11 catalytic mutant proteins generated using in vitro transcription/translation. The membrane was probed using anti-DUSP11 and anti-tubulin antibodies. ( B ) In vitro phosphatase reactions on the [γ-32P]-BLV-pre-miR-B5 mimic and [γ-32P]-BLV-miR-B5-5p miRNA mimic using CIP (positive control) or the in vitro translated DUSP11, DUSP11 catalytic mutant, or luciferase (negative control) from A . Reactions were fractionated on 15% PAGE/8 M urea, and RNAs were stained with EtBr. RNAs were then transferred to a membrane, exposed to a storage phosphor screen, and imaged on a Typhoon bimolecular imager. ( C ) Northern blot analysis from wild-type and DUSP11 knockout HEK293T cells transfected with a 5′ triphosphorylated BLV-B5 pre-miRNA mimic pretreated with (+) or without (−) RNA 5′ polyphosphatase. The blot was first probed for the 5p miRNA arm (green), stripped, and reprobed for the 3p arm (orange). Note that a lighter exposure for the input RNA is shown as compared with the RNA recovered from cells.

Article Snippet: Ten picomoles of each mimic was incubated in a 20-µL reaction (50 mM Tris, 10 mM KCl, 5 mM DTT, protease inhibitor [Roche], 1 U of SUPERase In RNase inhibitor [ThermoFisher]) that contained 3.3 µL of in vitro translated products (DUSP11-3xFlag, DUSP11-CM-3x-Flag, or the negative control luciferase) or 1 µg of CIP (New England Biolabs) for 1 h at 37°C.

Techniques: Expressing, Mutagenesis, Generated, In Vitro, Positive Control, Luciferase, Negative Control, Polyacrylamide Gel Electrophoresis, Staining, Northern Blot, Knock-Out, Transfection

Journal: eLife

Article Title: ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

doi: 10.7554/eLife.06547

Figure Lengend Snippet: ULK3 phosphorylation of ESCRT-III proteins. ( A ) 293T cells were co-transfected with vectors expressing Myc-ESCRT-III proteins and either empty vector, OSF-ULK3 WT, or OSF-ULK3 K44H. Cell lysates were electrophoresed on 10% Phos-tag gels and blotted for the designated ESCRT-III proteins to reveal lower mobility phosphoproteins (designated by ‘asterisks’). Treatment with CIP confirmed that the mobility changes resulted from phosphorylation (final three lanes in each blot). ( B ) Overlay of ESI/MS spectra showing the intact mass of Myc-IST1 co-immunoprecipitated with either OSF-ULK3 WT (red) or the catalytically inactive OSF-ULK3 K139R protein together with CIP treatment (black). Theoretical vs actual measured masses of phospho-IST1 species detected in this experiment are tabulated. DOI: http://dx.doi.org/10.7554/eLife.06547.030

Article Snippet: The OSF-ULK3 K139R/IST1 elution was additionally incubated with CIP (New England Biolabs, Ipswich, MA) at 37°C for 1 hr.

Techniques: Transfection, Expressing, Plasmid Preparation, Mass Spectrometry, Immunoprecipitation

Journal: Scientific Reports

Article Title: Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site

doi: 10.1038/srep00415

Figure Lengend Snippet: Schematic representation of the LV cloning strategy. There is a BamH I clone site at the 5'-end of the original vectors pcDNA3.1/V5-His-Snk/hPlk2, and pEGFP-N1, respectively. A BamH I clone site was inserted at the 3'-ends of EGFP and hPlk2 WT by SDM, respectively. Then, K111M, T239D and T239V mutants were created through SDM using hPlk2 WT gene as template. The inserts were digested with BamH I, and purified. At the same time, pWPI vector was digested by BamH I, and treated with CIP to protect the self-circularization of the vector DNA. Finally, LVs were cloned through ligation and transformation.

Article Snippet: Preparation of vector and insert DNA The bicistronic LV pWPI (Addgene plasmid 12254) was modified by creating a BamH I site at 3502 nt and replacing EGFP sequence with Neo, to form pWPI/Neo/BamH I. pWPI/Neo/BamH I DNA was digested with BamH I (NEW ENGLAND BioLabs), then divided into two aliquots, one aliquot of the vector DNA was used directly for ligation, and another aliquot was treated with CIP (NEW ENGLAND BioLabs) to remove the 5’-phosphate groups ( ) as follows: in a 300µl reaction, containing digested pWPI/Neo DNA (about 15 µg), 50 U CIP in 1 X NEBuffer 3, at 37°C water bath for 1 hours.

Techniques: Clone Assay, Purification, Plasmid Preparation, Ligation, Transformation Assay

Journal: The Plant Cell

Article Title: APURINIC/APYRIMIDINIC ENDONUCLEASE2 and ZINC FINGER DNA 3′-PHOSPHOESTERASE Play Overlapping Roles in the Maintenance of Epigenome and Genome Stability [OPEN]

doi: 10.1105/tpc.18.00287

Figure Lengend Snippet: APE2 Has Weak DNA 3′ Phosphatase Activity and Strong DNA 3′-5′ Exonuclease Activity in Vitro. (A) APE2 activity against DNA substrate with a 3′-P terminus. The wild-type or mutant form of APE2 proteins (10 nM) was incubated with 3′-P terminated DNA substrate (10 nM) for the indicated times. (B) APE2 activity against DNA substrate with a 3′-OH terminus. Reactions were performed as described in (A) with the exception that the 3′-P in the DNA substrate was replaced by a 3′-OH. (C) Stronger exonuclease activities of APE2. APE2 proteins at low concentrations (1 or 2 nM) were incubated with 3′-P terminated or 3′-OH terminated DNA substrate (10 nM) for 30 min. (D) Release of free phosphate residue from 3′- 32 P-labeled DNA substrate by APE2. APE2 proteins at different concentrations were incubated with 3′- 32 P-labeled DNA substrate (0.05 pM) for 3 h. CIP (5 and 10 units) was used as positive controls; np, no protein control. (E) Phenotypes of 30-d-old zdp-1ape2-2 seedlings complemented by wild-type or mutant forms of APE2. (F) Analysis of the DNA methylation level at the Chr2:17657100-17659549 locus by chop-PCR. Hap II is a methylation-sensitive restriction enzyme. DNA hypermethylation results in an increased level of the PCR product. Undigested DNA was amplified as a control.

Article Snippet: The DNA substrate with 3′-blocking phosphate (10 nM) was incubated with commercial CIP (NEB) or purified His-APE2 proteins in 1× CutSmart buffer containing 50 mM potassium acetate (pH 7.9), 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 μg/mL BSA (NEB) at 22°C for the indicated time.

Techniques: Activity Assay, In Vitro, Mutagenesis, Incubation, Labeling, DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Amplification