recombinant human clk1  (Carna Inc)

Journal: Cell Reports Medicine

Article Title: Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

doi: 10.1016/j.xcrm.2023.101354

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Article Snippet: TaqManTM Gene Expression Assay (FAM) Inventoried human CLK1 (Hs00964634) , Thermofisher Scientific , Cat# 4331182.

Techniques: Plasmid Preparation, Recombinant, Binding Assay, Staining, Red Blood Cell Lysis, DC Protein Assay, Gene Expression, DNA Purification, Purification, Reverse Transcription, Luminex, RNA Sequencing, Amplification, Oligonucleotide Synthesis, Software, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Electroporation, Multiplex Assay

Journal: Cell Reports Medicine

Article Title: Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

doi: 10.1016/j.xcrm.2023.101354

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Article Snippet: TaqManTM Gene Expression Assay (FAM) Inventoried human CLSPN (Hs08898637) , Thermofisher Scientific , Cat# 4331182.

Techniques: Plasmid Preparation, Recombinant, Binding Assay, Staining, Red Blood Cell Lysis, DC Protein Assay, Gene Expression, DNA Purification, Purification, Reverse Transcription, Luminex, RNA Sequencing, Amplification, Oligonucleotide Synthesis, Software, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Electroporation, Multiplex Assay

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 1 Depletion of SR kinases has differential effects on HIV-1 protein levels. a Schematic of HIV-1 rtTAGagzipGFP provirus used to generate CEM-HIV* cell line. b, c CEM-HIV* cells were infected with shRNA lentivirus targeting CLK1, CLK2, CLK3, or SRPK1 and transduced cells were selected with puromycin for 72 h. Following puromycin selection, HIV-1 gene expression was induced with doxycycline (Dox, 4.5 µM) + prostratin (Pros,2.56 µM) and cells harvested for western blots after 24 h of induction. Shown are the representative western blots indicating expression levels of b the target kinase, c or HIV-1 Env, Gag, and Tat levels. Band intensity was quantified relative to Dox induced shRNA control and normalized to either total protein stain for Env and Gag blots or GAPDH for Tat blots using Bio-Rad ImageLab software. Data are indicated as mean ± SEM, n ≥ 4 independent experiments, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. Dotted vertical lines on the blots represent cropping of lanes on the same representative blot to show shcontrol lanes adjacent to shRNA target depletion lanes

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Infection, shRNA, Selection, Gene Expression, Western Blot, Expressing, Control, Staining, Software

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 2 Effect of SR kinase depletion on HIV-1 RNA accumulation. a Schematic of HIV-1 provirus indicating the position of primers used to detect viral RNAs. b CEM-HIV* cells were depleted of CLK1, CLK2, CLK3, or SRPK1 by transduction with lentiviruses expressing shRNAs to these SR kinases. Following selection of transduced cells with puromycin for 72 h, HIV-1 gene expression was induced by addition of Dox (4.5 µM) + prostratin (2.56 µM). Cells were harvested for RNA extraction after 24 h of induction. HIV-1 unspliced (US), singly spliced (SS), multiply spliced (MS) RNA levels were determined by RT-qPCR. Viral mRNA levels were normalized to ß-actin and the mean mRNA levels expressed relative to sh control. Data are indicated as mean ± SEM, n = 3 independent experiments, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Transduction, Expressing, Selection, Gene Expression, RNA Extraction, Quantitative RT-PCR, Control

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 3 CLK1 but not CLK2/3 depletion alters HIV-1 transcription initiation and enhances response to LRAs. a Quantification of TAR and R-U5-Gag RNA levels in CEM-HIV* cells depleted of individual CLK1, 2, 3, or SRPK1 by shRNA lentivirus. Relative quantification was performed using comparative cycle threshold (CT) values. PUM1 was used as a reference gene to normalize the CT value and the fold changes calculated using 2−

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: shRNA, Quantitative Proteomics

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 4 Activation of primary CD4+ T cells selectively alters SR kinase and SR protein levels. Primary CD4+ T cells were isolated from healthy (HIV uninfected) human donors and untreated (control) or treated with activators (anti-CD3/CD28 and IL-2). Cells were harvested at different times (24 h, 48 h, 4 d, and 6 d) with or without activation for analyses by western blots or RT-qPCR to look for changes in the expression of SR kinases and SR proteins. a Top and bottom panels on the left are the representative western blots probed for CLK1, CLK2, CLK3, and SRPK1. Top and bottom panels on the right are the quantitation of blots for at least 3 donors (except for 4 d and 6 d post-activation for CLK2, CLK3, and SRPK1 expression levels where only one donor was used). b Quantification of CLK1 and SRPK1 mRNA levels in CD4+ T cells of 3 donors by RT-qPCR assay. mRNA levels were normalized to ß2-microglobulin and mean mRNA levels were expressed relative to untreated control. c Quantitation of western blots for SR protein expression levels in untreated versus treated/activated CD4+ T cell lysates (see Additional file 1: Fig. S4 for representative western blots) across at least 3 donors. For western blots, band intensity was quantified relative to untreated control and normalized to total protein load using Bio-Rad ImageLab software. Data are indicated as mean ± SD, n = 3 or 4 independent experiments, *p ≤ 0.05 and **p ≤ 0.01

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Activation Assay, Isolation, Control, Western Blot, Quantitative RT-PCR, Expressing, Quantitation Assay, Software

Journal: Retrovirology

Article Title: Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.

doi: 10.1186/s12977-022-00605-4

Figure Lengend Snippet: Fig. 8 CLK1 and CLK2 act at distinct steps to regulate HIV-1 gene expression. HIV-1 provirus integrated into the host genome is transcribed by the cellular RNA polymerase II (RNAP II). CLK1 acts to suppress the use of the HIV-1 promoter while CLK2 promotes steps in viral RNA synthesis/ processing post initiation (elongation, splicing, or 3′ end formation)

Article Snippet: Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl2, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 μM of ATP (Promega, Madison, WI, USA).

Techniques: Gene Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Induction of beige‐like adipocyte markers and functions in 3T3‐L1 cells by Clk1 and PKCβII inhibitory molecules

doi: 10.1111/jcmm.17345

Figure Lengend Snippet: SiRNA reduction of Clk 1, 2 and 4 levels by specific siRNA reveals that Clk1 inhibition regulates levels of UCP1 and PGC1α. (A) siRNA to Clk1 reduced Clk1 levels by 50% while UCP1 mRNA levels increased fourfold, and PGC1α increased 3.5‐fold. (B) siRNA to Clk2 reduced Clk2 levels by 40%, but only increased UCP1 mRNA by 20% while PGC1α was only increased 50%. (C) siRNA to Clk4 reduced Clk4 by 70%; it reduced UCP1 mRNA by 70%, and PGC1α was increased only 15%. A–C. TG003 (50 nM) treatment consistently increased UCP1 by 3–4 fold, PGC1α by twofold, and had minimal effects on lowering Clk levels. (** p < 0.01 for siClk1,2 and 4 or TG003 vs. scrambled siRNA which was equivalent to control in all cases as evaluated using ANOVA). (D) Oxygen Consumption Rates (OCR) for untreated and TG003‐treated 3T3‐L1 adipocytes. Basal OCR increased 25 pmol/min in TG003‐treated cells vs. control. Proton leak was increased 2.3‐fold with TG003 treatment. Spare receptor capacity increased 50 pmol/min while ATP production was reduced approximately 45% in TG003‐treated cells. Data shown are mean ± SEM (** p < 0.01 for 5 separate experiments performed in duplicate and analysed by Friedman's paired t ‐test)

Article Snippet: Clk1 siRNAs (IDs: SR300856C), Clk2 siRNAs (IDs:SR416008A‐C) and Clk4 siRNAs (IDs: SR408785A‐C) along with scrambled control siRNA was purchased from Origene, and transfected using Origene's siTran transfection reagent.

Techniques: Inhibition, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Induction of beige‐like adipocyte markers and functions in 3T3‐L1 cells by Clk1 and PKCβII inhibitory molecules

doi: 10.1111/jcmm.17345

Figure Lengend Snippet: Binding of TG003 and PGC1α to Clk 1. (A) Steady state SPR of ~1800 RU Clk1 affixed to an Ni‐NTA chip and titrated with TG003 in HBS‐N with 1% DMSO plotted in GraphPad Prism using an one‐site specific binding model with a K D of 25.24 with a 95% confidence interval of 12.93 to 48.53 nM. (B) Steady state SPR of ~400 RU Clk1, ~1000 RU of Clk4 and ~1000 RU Clk2 attached to a CM5 chip via NHS/EDC chemistry indicating binding of Clk1 and Clk4 to TG003 greater than binding of Clk2 to TG003. (C) Steady state SPR of Clk1, Clk2 and Clk4 as in 7B, indicating affinity of Clk1 to PGC1α of 240 nM +/− 28 nM, Clk2 to PGC1α of 980 nM +/− 89 nM, and Clk4 to PGC1α of 509 nM +/− 73 nM. (D) Ligand interaction diagram of TG003 ⁵ bound to Clk1 (PDB ID: 1Z57) generating grid from ligand in crystal structure. (E) 100 ns simulation of Clk1 in the absence of ligand prepared using Schrodinger Protein Preparation Wizard on PDB ID: 1Z57 and NAMD run using CHARMM‐GUI Solution Builder with RMSD of aligned backbone taken over the 1000 frames of the trajectory with each frame equal to 100 ps. (F) ClusPro predicted binding of PGC1α fragment from (PDB ID: 5UNJ) minimized with Protein Preparation Wizard and bound to receptor Clk1 from 7D aligned and one CLK deleted indicating proximal binding to nearby region of Clk1. (G) Heatmap to visualize inhibition of kinases by TG003

Article Snippet: Clk1 siRNAs (IDs: SR300856C), Clk2 siRNAs (IDs:SR416008A‐C) and Clk4 siRNAs (IDs: SR408785A‐C) along with scrambled control siRNA was purchased from Origene, and transfected using Origene's siTran transfection reagent.

Techniques: Binding Assay, Inhibition

Journal: Nature microbiology

Article Title: Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors

doi: 10.1038/s41564-020-0745-6

Figure Lengend Snippet: (a) Localization of kinetochore protein KKT2 after CLK1 inhibition by AB1. Parasites were incubated or not for 24 h with 2x EC 50 (upper panel) or 6 h with 5 x EC 50 AB1 (lower panel). Representative fluorescence micrographs, showing bloodstream form parasites endogenously expressing N-terminal mNeonGreen (mNG) tagged KKT2. Cells in metaphase and anaphase are shown. Cells were counterstained with DAPI to visualize DNA (cyan). The right panel shows the Nomarsky (DIC) corresponding images. Scale bar, 2μm. Upper right panel shows cell cycle progression after treatment with 2x EC 50 AB1 for 72 h. Data are representative from one of three independent biological replicates with similar results. (b) Localization of KKT2 after CLK1 depletion by RNAi. Representative fluorescence micrographs, showing 24 h induction of CLK1 RNAi in bloodstream form parasites endogenously expressing N-terminal mNeonGreen (mNG) labelled KKT2, compared with not induced control cells in metaphase and anaphase are shown. Cells were counterstained with DAPI to visualize DNA (cyan). The right panel shows the Nomarsky (DIC) corresponding images. Scale bar, 2μm. Data are representative from one of three independent biological replicates with similar results.

Article Snippet: Human CLK1 (hCLK1; P49759) was obtained from Promega.

Techniques: Inhibition, Incubation, Fluorescence, Expressing, Control

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 and TG003 chemical structures. ( b ) Pharmacokinetic profile of TG693 after a single 30 mg kg −1 dose administered by subcutaneous injection in imprinting control region (ICR) mice. Data indicate the mean ± SEM (n = 3). ( c ) Recombinant CLK1 was incubated with the substrate peptide in the presence of the indicated concentrations of small molecules. Data represent the means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) TG693 competitive ATP inhibition is shown in Michaelis-Menten (left) and Hanes-Woolf (right) plots. CLK1 kinase activity was measured at the indicated concentrations of TG693 and ATP. Velocity was plotted versus [ATP] and [ATP]/velocity was plotted versus [ATP]. ( e ) Map of the inhibitory activities of TG693 on a kinase dendrogram. Percent inhibition by 1 μM TG693 was measured for a panel of 313 kinases. Red circles indicate the inhibited kinases and are sized according to percent inhibition. The illustration was reproduced courtesy of Cell Signaling Technology, Inc. ( www.cellsignal.com ).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Injection, Recombinant, Incubation, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) SR protein phosphorylation was assessed in HeLa cells treated with TG693 and TG003 for 1 h. Lamin B served as a loading control. Uncropped images have been provided in . ( b , c ) Effect of TG693 on exon 31 skipping with the reporter plasmid. Transfected HeLa cells were incubated in the presence of TG693, TG003, or DMSO vehicle for 24 h. Reporter and endogenous CLK1 splicing was then analyzed by RT-PCR. GAPDH served as a control. The Splicing ratios were quantified by intensity analysis and normalized to GAPDH expression. Uncropped images have been provided in and , respectively. Data represent the means ± SD (n = 3).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Plasmid Preparation, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 bioavailability in the tibialis anterior (TA) muscle of ICR mice after oral administration of a single 30 mg kg −1 dose. Data represent the mean ± SEM (n = 3). ( b ) SR protein phosphorylation status in the TA muscle of ICR mice after oral administration. Lamin B served as a loading control. SRSF4 phosphorylation was quantified by densitometry. Uncropped images are provided in . Data represent means ± SD (n = 5). * p < 0.05. ( c,d ) Clk1 expression in the TA muscle, heart and diaphragm were analyzed by RT-PCR with a GAPDH internal control. Uncropped images are provided in and in , respectively. Data represent the means ± SD (n = 3). * p < 0.05.

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction