m0290  (New England Biolabs)

Journal: Molecular and Cellular Biology

Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

doi: 10.1128/MCB.23.12.4139-4149.2003

Figure Lengend Snippet: Ser/Thr and Tyr autophosphorylation independently influence substrate specificity. Increasing amounts (8, 20, and 50 ng) of P-Clk/Sty (lanes 2 to 4), PTP-Clk/Sty (lanes 5 to 7), and CIP-Clk/Sty (lanes 8 to 10) were added to splicing reactions performed with S100 extracts in the presence of phosphatase inhibitors and ASF/SF2 (A) or SC35 (B). Reaction mixtures were deproteinized after 80 min, and RNAs were precipitated with ethanol. RNAs were fractionated by 5% denaturing PAGE and visualized by autoradiography.

Article Snippet: Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions.

Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography

Journal: Molecular and Cellular Biology

Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

doi: 10.1128/MCB.23.12.4139-4149.2003

Figure Lengend Snippet: Autophosphorylation of Clk/Sty modulates kinase activity towards ASF/SF2 but not SRp40. (A) Increasing amounts of P-Clk/Sty (1, 5, and 20 ng) and equivalent amounts of PTP- and CIP-Clk/Sty were incubated with 1 μg of MBP under kinase conditions. Reactions were stopped by adding 1/2 volume of 3× SDS sample buffer, and proteins were fractionated by 10% SDS-PAGE and detected by autoradiography. (B and C) Increasing amounts of P-Clk/Sty (5, 20, and 100 ng) or equivalent amounts of PTP- and CIP-Clk/Sty were added to 1 μg of ASF/SF2 (B) or SRp40 (C) purified from E. coli and incubated for 30 min at 37°C. Reactions were processed as described for panel A except that proteins were transferred to nitrocellulose following PAGE. The extent of γ- 32 P incorporation was visualized by autoradiography (lanes 1 to 10). Subsequently, blots were incubated with the monoclonal antibody mAb104 (lanes 11 to 20) and reactivity was detected by chemiluminescence.

Article Snippet: Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions.

Techniques: Activity Assay, Incubation, SDS Page, Autoradiography, Purification, Polyacrylamide Gel Electrophoresis

Journal: Molecular and Cellular Biology

Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

doi: 10.1128/MCB.23.12.4139-4149.2003

Figure Lengend Snippet: Clk/Sty is autophosphorylated on Ser/Thr and Thr. (A) A total of 1 μg each of GST-Clk/Sty (lane 1) and a kinase-inactive mutant (GST-ClkR/Sty; lane 2) was fractionated by 10% SDS-PAGE and stained with Coomassie blue. The species indicated by the asterisk reflects an N-terminal truncation consisting of GST plus an approximately 10-kDa segment of Clk/Sty, which became phosphorylated in the wild-type but not the mutant sample. (B) A total of 0.5 μg of P-, PTP-, and CIP-Clk/Sty (lanes 1 to 3, respectively), was fractionated by 10% SDS-PAGE and stained with Coomassie blue. (C) A total of 20 to 50 ng of P-Clk/Sty (lane 1), PTP-Clk/Sty (lane 2), and CIP-Clk/Sty (lane 3) was fractionated by 10% SDS-PAGE and transferred to nitrocellulose. The blot was probed with antiphosphotyrosine antibody, and proteins were detected by chemiluminescence.

Article Snippet: Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions.

Techniques: Mutagenesis, SDS Page, Staining

Journal: The Plant Cell

Article Title: Biochemical and Genetic Requirements for Function of the Immune Response Regulator BOTRYTIS-INDUCED KINASE1 in Plant Growth, Ethylene Signaling, and PAMP-Triggered Immunity in Arabidopsis [C] [C] [W]

doi: 10.1105/tpc.111.087122

Figure Lengend Snippet: BIK1 Conserved Residues and in Vitro and in Vivo Kinase Assays. (A) Structure of the BIK1 protein and comparison of residues in the ADs of BIK1 and related kinases. Gray region denotes the KD and black the AD. Residues noted in the BIK1 protein indicate those substituted for in planta assays. (B) Kinase activity of recombinant BIK1 and Ala substitution mutants produced in E. coli detected by autoradiogram. CCB, Coomassie blue staining. (C) and (D) BIK1 substitution mutants in vivo detected by a mobility shift on an HA-immunoblot or by phosphoserine/Thr-specific antibody. (E) BIK1 and MBP phosphorylation is abrogated by phosphatase treatment. Protein dephosphorylation was performed according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units CIP/μg protein (left) or (~2.5 μ/μg) (right). −, Buffer; +, CIP. In (C) and (D) , plants were treated with ACC (Ac) or flg22 (Fl) for 3 h and assayed for changes in BIK1 and MBP phosphorylation activity. In (C) to (E) , in vivo BIK1 phosphorylation was detected by ACC or flagellin-induced mobility shifts observable by HA-immunoblot. The top band corresponds to phosphorylated BIK1, which is migrating slower than the unphosphorylated form. MBP phosphorylation by BIK1 was detected by immunoblots with a phosphoserine/Thr-specific antibody.

Article Snippet: Protein dephosphorylation was performed using calf intestinal alkaline phosphatase (CIP) according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units of CIP (as indicated)/μg protein.

Techniques: In Vitro, In Vivo, Activity Assay, Recombinant, Produced, Staining, Mobility Shift, De-Phosphorylation Assay, Western Blot

Journal: Journal of Virology

Article Title: Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis

doi: 10.1128/JVI.00807-18

Figure Lengend Snippet: Characterization of the anti-EF-1δ-S133 P monoclonal antibody. (A) HEK293T cells were transfected with a plasmid expressing EGFP-EF-1δ(F) (lane 1 and 2) or a plasmid expressing EGFP-EF-1δS133A(F) (lane 3) and harvested at 48 h posttransfection. Cell lysates were mock treated (lanes 1 and 3) or treated with CIP (lane 2) and then analyzed by immunoblotting with anti-Flag monoclonal antibody (top), anti-EF-1δ-S133 P monoclonal antibody (middle), or anti-β-actin monoclonal antibody (bottom). β-Actin was used as a loading control. (B) U2OS cells were mock infected (lane 1) or infected with wild-type HSV-2 186 (lane 2), YK862 (ΔUL13) (lane 3), or YK863 (ΔUL13-repair) (lane 4) at an MOI of 3 and harvested at 24 h postinfection, and lysates were analyzed by immunoblotting with the indicated antibodies. α-Tubulin and UL37 were used as a loading control and an infection indicator, respectively. (C) U2OS cells were mock infected (lanes 1) or infected with wild-type HSV-2 186 (lane 2), YK864 (UL13-K176M) (lane 3), or YK865 (UL13-K176M-repair) (lane 4) and harvested, and lysates were analyzed as described above for panel B. Molecular mass markers are shown on the left.

Article Snippet: Lysates of Vero cells that had been infected with wild-type HSV-2 186 at an MOI of 3 for 24 h and lysates of HEK293T cells that had been transfected with pEGFP-EF-1δ(F) were treated with calf intestinal alkaline phosphatase (CIP) (New England BioLabs) as described previously ( ).

Techniques: Transfection, Plasmid Preparation, Expressing, Infection

Journal: Non-Coding RNA

Article Title: “Pocket-sized RNA-Seq”: A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest

doi: 10.3390/ncrna1020127

Figure Lengend Snippet: Scheme of the pocket-sized RNA-seq method. 5P-, 5′-phosphate group; 3OH-, 3′-hydroxyl group; 5-, dephosphorylated 5′-end; (AAA)n-3OH, polyadenylated 3′-end; (AAA)n and (TTT)n, polyA and polyT sequences, respectively; RACE, Rapid amplification of cDNA ends; RT, reverse transcription; CIP, calf intestinal phosphatase; oligo-biot, oligonucleotide biotinylated in 5′-end.

Article Snippet: One microgram of bait RNA was therefore dephosphorylated using 1 U CIP (Alkaline Phosphatase, Calf Intestinal, New England Biolabs, Evry, France) at 37 °C for 60 min and RNAs were purified by phenol-chloroform extraction.

Techniques: RNA Sequencing Assay, Rapid Amplification of cDNA Ends