Journal: PLoS ONE

Article Title: Phosphorylation of ERK5 on Thr732 Is Associated with ERK5 Nuclear Localization and ERK5-Dependent Transcription

doi: 10.1371/journal.pone.0117914

Figure Lengend Snippet: (A) HEK293 cells were co-transfected with a constitutively active MEK5 mutant (MEK5D) and mycERK5 wild type (WT) or mycERK5 kinase-dead (KD) mutant. Empty vector (Vec) was used as a control. Cells were lysed, and ERK5 phosphorylation status at the TEY site, Thr732 and Ser769/Ser773/Ser775, as well as mycERK5, ERK1/2 phosphorylation at TEY, and ERK2 were examined by Western blotting. (B) HEK293 cells were co-transfected with MEK5D (or Vec) and mycERK5-WT, mycER–32A or mycERK5-KD. ERK5 phosphorylation status at the TEY site and Thr732, and mycERK5 were examined by Western blotting. (C) HEK293 cells were co-transfected with MEK5D (or Vec) (upper panels), constitutively active RAS mutant (RASV12) (or Vec) (lower panels) and mycERK5 (upper) or mycERK2 (lower). U0126 (30 μM) was added, and the phosphorylation status of ERK5 and ERK2 at their TEY sites and mycERK5 or mycERK2 were examined by Western blotting.

Article Snippet: GST-tagged ERK5 and ERK2 recombinant proteins were purchased from SignalChem (Richmond, Canada).

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Phosphorylation of ERK5 on Thr732 Is Associated with ERK5 Nuclear Localization and ERK5-Dependent Transcription

doi: 10.1371/journal.pone.0117914

Figure Lengend Snippet: (A) PC12 cells were lysed, and endogenous ERK2 was immunoprecipitated using an anti-ERK2 antibody. Endogenous ERK5 and ERK2 in the immunoprecipitates or total cell lysate (TCL) were examined by Western blotting. (B) PC12 cells were stimulated with NGF (100 ng/ml) for 5 min, then the cells were lysed, and endogenous ERK2 was immunoprecipitated using an anti-ERK2 antibody. Endogenous ERK5 and ERK2 in the immunoprecipitates or total cell lysate (TCL) were examined by Western blotting.

Article Snippet: GST-tagged ERK5 and ERK2 recombinant proteins were purchased from SignalChem (Richmond, Canada).

Techniques: Immunoprecipitation, Western Blot

Journal: International journal of cancer

Article Title: Constitutively active MEK1 is sufficient to induce epithelial-to-mesenchymal transition in intestinal epithelial cells and to promote tumor invasion and metastasis.

doi: 10.1002/ijc.24485

Figure Lengend Snippet: FIGURE 1 – Constitutive active MEK1 induces a loss of epithelial morphology and inhibition of epithelial junctional proteins. (a) Left panel. Cell extracts (200 lg) were immunoprecipitated with a specific antibody to HA. Levels of immunoprecipitated HA-wtMEK or HA-caMEK were analyzed by Western blotting. Kinase activity of HA-wtMEK and HA-caMEK is demonstrated by the phosphorylation of recombinant ERK2 revealed by Western blot with the antibody recognizing phosphorylated forms of ERK1/2. -: no cell lysate was added in the assay. Right panel: Cell lysates were prepared from asynchronously growing cells stably expressing pLXIN, wtMEK and caMEK. caMEK-expressing cells were incubated for 2 hr with DMSO or 20 lM U0126. Equal amounts of proteins were separated by SDS-PAGE for Western blot analysis of HA tag, phosphorylated and total ERK proteins; arrowhead indicates an additional species with low electrophoretic mobility that could correspond to ERK1b. (b) Representative phase/contrast pictures of subconfluent pLXIN-, wtMEK- and caMEK-expressing cells (panels 1–3). In panel 4, caMEK-expressing cells were treated with 20 lM U0126 during 72 hr. Bars: 50 lM. (c) pLXIN-, wtMEK- and caMEK-expressing cells were fixed for immunofluorescence and stained for E-cadherin (panels 1–3), ZO-1 (panels 4–6) or F-actin (panels 7–9) with FITC-phalloidin. Scale bars 50 lm. (d) Equal amounts of whole cell lysates from wtMEK- and caMEK-expressing cells (treated or untreated with 20 lM U0126 during 72 hr) were separated by 7.5 or 10% SDS-PAGE, and proteins were analyzed by Western blotting with specific antibodies against E-cadherin, occludin-1, ZO-1, N-cadherin, Vimentin, b-actin and b-tubulin.

Article Snippet: The recombinant ERK2 protein was purchased from Upstate Biotechnology (Lake Placid, NY).

Techniques: Inhibition, Immunoprecipitation, Western Blot, Activity Assay, Phospho-proteomics, Recombinant, Stable Transfection, Expressing, Incubation, SDS Page, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Formation of E-cadherin-mediated cell-cell adhesion activates AKT and mitogen activated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovarian cancer cells.

doi: 10.1210/me.2004-0342

Figure Lengend Snippet: Fig. 2. E-Cadherin-Mediated Cellular Adhesion Induces Akt and MAPK Activation A, Starved OVCAR-3 cell monolayers were left untreated (Control) or treated with 4 mM EGTA for 30 min (EGTA 30 min). The EGTA-containing medium was then replaced with serum-free, calcium-containing (1.8 mM) medium for 5 min (E-Cad Restoring Calcium 5 min). Afterward, the cells were fixed, permeabilized, and stained with anti-E-cadherin, anti--catenin, and anti-ZO-1 antibodies followed by fluorescein isothiocyanate-conjugated secondary antibodies and prepared for microscopic analysis at a magnification of 63. B, OVCAR-3 cells were starved of serum overnight and treated with 4 mM EGTA for 30 min and subsequently restored with calcium-containing (1.8 mM) serum-free medium for 5–90 min, as indicated. As a control, starved OVCAR-3 cells were left untreated, treated with EGTA only, or with 30% fetal bovine serum (FBS) for 30 min. After calcium restoration, cell lysates were blotted with anti-phospho-Akt (Ser473) (P-Akt) and anti-phospho-p44/42 MAPK (Thr202/Tyr204) (P-MAPK). Amounts of similar proteins loaded were monitored using blots with anti-Akt (Total Akt) and anti-p44/42 MAPK (Total MAPK). The Western blot shows a representative result from three independent experiments, and normalized values (mean SD) from three independent experiments for changes in levels of P-Akt and P-MAPK were also shown. C, Phosphorylated Akt was immunoprecipitated from cell lysates and an Akt kinase assay was performed using recombinant GSK as Akt substrate, as described in Materials and Methods. Both Akt kinase activities (Akt activity) and the amount of immunoprecipitated phosphor- ylated Akt (IP-P-Akt) are shown. Normalized values (mean SD) from three independent experiments for changes in Akt kinase activities were also shown. Lowercase letters (a, b, c, and d) indicate significant differences (P 0.05).

Article Snippet: Rabbit polyclonal antibodies against p44/42 MAPKs (MAPK, or ERK 1 and 2), phosphorylated p44/42 MAPK (Thr202/Tyr204, p-MAPK), Akt (PKB), phosphorylated Akt (Ser473), and Gab 1 were from Cell Signaling Technologies (Beverly, MA).

Techniques: Activation Assay, Control, Staining, Western Blot, Immunoprecipitation, Kinase Assay, Recombinant, Activity Assay

Journal: eLife

Article Title: Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes

doi: 10.7554/eLife.65512

Figure Lengend Snippet:

Article Snippet: Antibody , p44/42 MAPK (Erk1/2) (Total Erk) (Rabbit polyclonal) , Cell Signaling Technology , 9102 , WB (1:1000).

Techniques: Control, Recombinant, Flow Cytometry, Imaging, Plasmid Preparation, Software, Sequencing

Journal: Oncogenesis

Article Title: Phosphorylation of 14-3-3ζ links YAP transcriptional activation to hypoxic glycolysis for tumorigenesis

doi: 10.1038/s41389-019-0143-1

Figure Lengend Snippet: a Coimmunoprecipitation assay assessing the interaction between 14-3-3ζ and YAP in SW-1990 PDAC cells stimulated with hypoxia in the presence or absence of 5 μmol/L BAY 11-7085 (BAY) and 20 μmol/L U0126 pretreatment. Data are expressed as mean ± s.d. of three independent experiments. * P < 0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. b Left panel: Western-blotting assessing the levels of ERK2 expression in SW-1990 PDAC cells transfected with ERK2 siRNA. Data are expressed as mean ± s.d. of three independent experiments. ** P < 0.01. Two-sided Student’s t test was used to calculate the P value. Right panel: Coimmunoprecipitation assay detecting the interaction between 14-3-3ζ and YAP in SW-1990 PDAC cells stimulated with hypoxia in the presence or absence of ERK2 siRNA transfection. Data are expressed as mean ± s.d. of three independent experiments. ** P < 0.01. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. c In vitro protein binding assay with mixing recombinant ERK2 (rERK2) kinase and IPs of e14-3-3ζ from SW-1990 PDAC cells followed by WB analyses with an anti-YAP antibody. Data are expressed as mean ± s.d. of three independent experiments. * P < 0.05. Two-sided Student’s t test was used to calculate the P value. d Subcellular fractionation analyses assessing nuclear YAP localization in hypoxia-stimulated SW-1990 PDAC cells with 5 μmol/L BAY 11-7085 (BAY) and 20 μmol/L U0126 pretreatment, respectively. e Representative immunfluorescence images of nuclear YAP localization in hypoxia-stimulated SW-1990 PDAC cells with 5 μmol/L BAY 11-7085 (BAY) and 20 μmol/L U0126 pretreatment, respectively, and in hypoxia-stimulated SW-1990 PDAC cells with or without ERK2 siRNA transfection. Scale bar = 25 μm. f Subcellular fractionation analyses comparing nuclear YAP localization in hypoxia-stimulated SW-1990 PDAC cells with or without ERK2 siRNA transfection

Article Snippet: The antibodies against 14-3-3ζ (monoclonal, sc-293415), ERK2 (monoclonal, sc-136288) and WWTR1 (monoclonal, sc-293183) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Expressing, Transfection, In Vitro, Protein Binding, Recombinant, Fractionation

Journal: Oncogenesis

Article Title: Phosphorylation of 14-3-3ζ links YAP transcriptional activation to hypoxic glycolysis for tumorigenesis

doi: 10.1038/s41389-019-0143-1

Figure Lengend Snippet: a Coimmunoprecipitation assay determining the interaction between endogenous 14-3-3ζ and ERK1/2 in SW-1990 PDAC cells stimulated with hypoxia for 6 h. Data are expressed as mean ± s.d. of three independent experiments. * P < 0.05. Two-sided Student’s t test was used to calculate the P value. b GST pull-down assay examining the interaction between purified GST-ERK2 proteins and IPs of Flag-tagged wild-type 14-3-3ζ from hypoxia-stimulated SW-1990 PDAC cells. c Recognition D-site motif of 14-3-3ζ and other known ERK2 substrates for comparison. d Coimmunoprecipitation assay determining the interaction between Flag-tagged wild-type 14-3-3ζ or mutant 14-3-3ζ L98/100 A and ERK1/2 in SW-1990 PDAC cells stimulated with hypoxia for 6 h. e Structural model of the ERK2 (PDB ID: 4H3Q) and 14-3-3ζ (PDB ID: 1IB1) complex. The interacting residues involved in the interface between ERK2 and D-site peptide of mitogen-activated protein kinase (MAPK) docking domain at 14-3-3ζ are as indicated. f Coimmunoprecipitation assay assessing the abundance of Flag-tagged wild-type 14-3-3ζ phosphorylation in hypoxia-stimulated SW-1990 PDAC cells in the presence or absence of ERK2 siRNA transfection with an anti-phospho-serine antibody. g Coimmunoprecipitation assay determining the levels of Flag-tagged wild-type 14-3-3ζ (WT) and mutant 14-3-3ζ S37A (S37A) phosphorylation in hypoxia-stimulated SW-1990 PDAC cells with an anti-14-3-3ζ pS37 antibody. h In vitro protein kinase assay for detection of 14-3-3ζ Ser37 phosphorylation with mixing recombinant ERK2 protein (rERK2) and IPs of Flag-tagged wild-type 14-3-3ζ (WT) or mutant 14-3-3ζ S37A (S37A) followed by WB analyses with an anti-14-3-3ζ pS37 antibody. Asterisk indicates the non-specific bands

Article Snippet: The antibodies against 14-3-3ζ (monoclonal, sc-293415), ERK2 (monoclonal, sc-136288) and WWTR1 (monoclonal, sc-293183) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Purification, Comparison, Mutagenesis, Phospho-proteomics, Transfection, In Vitro, Protein Kinase Assay, Recombinant

Journal: Oncogenesis

Article Title: Phosphorylation of 14-3-3ζ links YAP transcriptional activation to hypoxic glycolysis for tumorigenesis

doi: 10.1038/s41389-019-0143-1

Figure Lengend Snippet: a Coimmunoprecipitation assay evaluating the interaction of Flag-tagged wild-type 14-3-3ζ (WT) or mutant 14-3-3ζ S37A (S37A) with YAP in hypoxia-stimulated SW-1990 PDAC cells using an anti-YAP antibody. Data are expressed as mean ± s.d. of three independent experiments. * P < 0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. b In vitro protein binding assay for evaluation of 14-3-3ζ-YAP interaction with mixing purified His-tagged YAP immobilized on Ni 2+ -nitrilotriacetic acid (NTA)-sepharose beads and IPs of Flag-tagged wild-type 14-3-3ζ (WT), mutant 14-3-3ζ S37A (S37A) in the presence or absence of recombinant ERK2 protein (rERK2) followed by WB analyses with an anti-Flag antibody. PD, pull-down. c Subcellular fractionation analyses comparing nuclear YAP localization in 14-3-3ζ shRNA (sh.14-3-3ζ)-expressed SW-1990 PDAC cells with Flag-tagged wild-type 14-3-3ζ (WT), mutant 14-3-3ζ S37A (S37A) or S37E (S37E) reconstitution under normoxia and hypoxia. d Representative immunfluorescence images of nuclear YAP localization in 14-3-3ζ shRNA (sh.14-3-3ζ)-expressed SW-1990 PDAC cells with Flag-tagged wild-type 14-3-3ζ (WT), mutant 14-3-3ζ S37A (S37A) or S37E (S37E) reconstitution under normoxia and hypoxia. Scale bar = 25 μm. e Coimmunoprecipitation assay determining the interaction between nuclear YAP and HIF-1α in 14-3-3ζ shRNA (sh.14-3-3ζ)-expressed SW-1990 PDAC cells with Flag-tagged wild-type 14-3-3ζ (WT) or mutant 14-3-3ζ S37A (S37A) reconstitution under hypoxia. f ChIP analysis for YAP binding to PKM2 gene HRE in 14-3-3ζ shRNA (sh.14-3-3ζ)-expressed SW-1990 PDAC cells with hypoxia stimulation in the presence or absence of Flag-tagged wild-type 14-3-3ζ (WT) or mutant 14-3-3ζ S37A (S37A) reconstitution using the indicated antibodies. g Western-blotting examining abundance of Flag and PKM2 protein expression in 14-3-3ζ shRNA (sh.14-3-3ζ)-expressed SW-1990 PDAC cells with hypoxia stimulation in the presence or absence of Flag-tagged wild-type 14-3-3ζ (WT) or mutant 14-3-3ζ S37A (S37A) reconstitution. h Luciferase-reporter PKM2 HRE activity analysis of 14-3-3ζ shRNA (sh.14-3-3ζ)-expressed SW-1990 PDAC cells with hypoxia stimulation in the presence or absence of Flag-tagged wild-type 14-3-3ζ (WT) or mutant 14-3-3ζ S37A (S37A) reconstitution. Data are expressed as mean ± s.d. * P < 0.05. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. i RT-qPCR analyses of PKM2 gene expression in 14-3-3ζ shRNA (sh.14-3-3ζ)-expressed SW-1990 PDAC cells with hypoxia stimulation in the presence or absence of Flag-tagged wild-type 14-3-3ζ (WT) or mutant 14-3-3ζ S37A (S37A) reconstitution. Experiments were performed five times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. * P < 0.05. Two-sided Student’s t test was used to calculate the P value

Article Snippet: The antibodies against 14-3-3ζ (monoclonal, sc-293415), ERK2 (monoclonal, sc-136288) and WWTR1 (monoclonal, sc-293183) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, In Vitro, Protein Binding, Purification, Recombinant, Fractionation, shRNA, Binding Assay, Western Blot, Expressing, Luciferase, Activity Assay, Quantitative RT-PCR, Gene Expression

Journal: Oncogenesis

Article Title: Phosphorylation of 14-3-3ζ links YAP transcriptional activation to hypoxic glycolysis for tumorigenesis

doi: 10.1038/s41389-019-0143-1

Figure Lengend Snippet: a Representative cases stained by immunohistochemistry (top panel) and bar graph (bottom panel) showing the expression of 14-3-3ζ p-S37 in 87 primary human PDAC specimens is positively correlated with the levels of p-ERK1/2 and HIF-1α. Scale bar = 50 μm. The P value shown was calculated by Spearman order correlations. b Kaplan-Meier curves comparing the overall survival (OS) and recurrence free survival (RFS) in 87 PDAC patients with low and high expression of 14-3-3ζ p-S37. Log-rank test was used to calculate the P value. c Proposed schematic illustrating a pivotal role for the ERK2-induced 14-3-3ζ Ser37 phosphorylation in promoting nuclear YAP localization and driving PKM2 transcription via physical YAP-HIF-1α interaction in response to hypoxic stress

Article Snippet: The antibodies against 14-3-3ζ (monoclonal, sc-293415), ERK2 (monoclonal, sc-136288) and WWTR1 (monoclonal, sc-293183) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Staining, Immunohistochemistry, Expressing, Phospho-proteomics

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

Journal: eLife

Article Title: Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

doi: 10.7554/eLife.77696

Figure Lengend Snippet: ( A ) Chemical structure of C17. ( B ) IC 50 values of C17 against DYRK1A, DYRKIB, DYRK3, Haspin and MARK3. ( C ) Kinome profiling of C17 at 500 nM was carried out using 468 human kinases ( https://www.discoverx.com/ ). ( D ) C17 inhibits Rpt3-Thr25 phosphorylation. HEK293T cells stably expressing FLAG-DYRK2 were treated with the indicated concentrations of C17 for 1 hr. The cells were lysed, and immunoblotting was carried out with the indicated antibodies. Figure 2—source data 1. Raw data of C17 Kinome profiling list for . Figure 2—source data 2. Raw data of western blot for .

Article Snippet: peptide, recombinant protein , GST-MARK3 protein , Carna Biosciences , Cat#: 02–122 , .

Techniques: Stable Transfection, Expressing, Western Blot

Journal: eLife

Article Title: Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

doi: 10.7554/eLife.77696

Figure Lengend Snippet: ( A–E ) IC 50 of C17 on DYRK2, DYRK1A, DYRK1B, DYRK3, Haspin and MARK3. The IC 50 graph was plotted using GraphPad Prism 8.4.0 software. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means ± SD for triplicate reactions with similar results obtained in at least one other experiment.

Article Snippet: peptide, recombinant protein , GST-MARK3 protein , Carna Biosciences , Cat#: 02–122 , .

Techniques: Software, Activity Assay

Journal: eLife

Article Title: Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

doi: 10.7554/eLife.77696

Figure Lengend Snippet:

Article Snippet: peptide, recombinant protein , GST-MARK3 protein , Carna Biosciences , Cat#: 02–122 , .

Techniques: Recombinant, Plasmid Preparation, Generated, Modification, Transfection, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Kinase Assay, Software

Journal: iScience

Article Title: PIMT regulates hepatic gluconeogenesis in mice

doi: 10.1016/j.isci.2023.106120

Figure Lengend Snippet: PIMT is phosphorylated by PKA at Ser 656 (A) Sepharose bound GST fused PIMT-C was subjected to kinase reaction in the presence of HeLa nuclear lysate, constitutively active purified MAPKS (ERK1 and ERK2), or PKA. (B) Primary Hepatocytes infected with lentivirus expressing PIMT-V5 were exposed to Forskolin (10 μM) with or without H89 (20 μM) or RP (20 μM) for 1h and then subjected to IP with PKA substrate antibody followed by immunoblot with indicated antibodies. (C) Densitometric quantification of Figure 4 B. The phosphorylation signals were normalized with their corresponding input. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ∗∗∗∗p < 0.001 compared to DMSO treated cells, a p< 0.001 compared to Forskolin treated cells. (D) Mice were infected with lentivirus expressing PIMT-V5 (n = 3). Post 7 days of infection, mice were fasted for 8h and liver lysates were subjected to IP with PKA substrate antibody followed by immunoblots with anti-V5. (E) Densitometric quantification of Figure 4 D. The phosphorylation signals were normalized with the corresponding input signals. Numerical data are expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t -test (two-tailed) ∗∗∗ p < 0.005, versus the corresponding input. (F) Glutathione Sepharose beads bound GST-PIMT-C (W and mutants) were subjected to kinase assay with active and purified PKA. Double mutations at the PKA recognition site (RxxS, Ser 656 , and Ser 851 ) abolished the phosphorylation of PIMT. (G) Primary hepatocytes isolated from female mice infected with either PIMT (W) or PIMT S656A were treated with Forskolin for 4h and then subjected to IP with PKA substrate antibody followed by immunoblots with the defined antibodies (n = 4). (H) Densitometric quantification of Figure 4 G. The phosphorylation signals were normalized with the corresponding input signals. Numerical data are expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t -test (two-tailed) ∗∗∗ p < 0.005, versus the corresponding input. (I) C57BL/6 male mice were tail-vein injected with lentivirus expressing PIMT (wt or S656A) (n = 3). Post 7 days of injection, mice fasted for 8h. Liver lysates were subjected to IP with PKA substrate antibody followed by immunoblots with the defined antibodies. (J) Densitometric quantification of Figure 4 G. The phosphorylation signals were normalized with the corresponding input signals. Numerical data are expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t-test (two-tailed) ∗∗∗ p < 0.005, versus the corresponding input. (K) 8h fasted liver lysates subjected to IP with PKA substrate antibody followed by immunoblots with the defined antibodies (n = 3). (L) Densitometric quantification of Figure 4 I. The phosphorylation signals were normalized with the corresponding input signals. Statistical analysis was performed using unpaired Student’s t-test (two-tailed) ∗∗ p < 0.01, versus the corresponding input. (M) HepG2 cells were transfected with the pGL3-PEPCK promoter and PIMT (W or mutants) encoding constructs with or without PKAc. Post-transfection cells were lysed, and luciferase readout was measured. The values were normalized with corresponding Renilla luciferase activity and expressed relative to PEPCK-Luc (unphosphorylated) (column 1), set to 1. Data are representative of 5 independent experiments and expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. d p< 0.001 vs PECK-Luc (unphosphorylated) ∗∗∗∗ p < 0.001 compared to PEPCK-Luc with PKAc transfected cells. γ p< 0.05, α p < 0.005 compared to PEPCK-Luc + PIMT (W) without PKAc, D p< 0.001 compared to PEPCK-Luc + PIMT (W) + PKAc, PIMT-W: PIMT wild type, PIMT-A: PIMT S656A mutant, PIMT-D: PIMT S656D mutant.

Article Snippet: Recombinant Human Active ERK2 Protein , RnD Systems , 1230-KS-010.

Techniques: Purification, Infection, Expressing, Western Blot, Phospho-proteomics, Two Tailed Test, Kinase Assay, Isolation, Injection, Transfection, Construct, Luciferase, Activity Assay, Mutagenesis

Journal: iScience

Article Title: PIMT regulates hepatic gluconeogenesis in mice

doi: 10.1016/j.isci.2023.106120

Figure Lengend Snippet:

Article Snippet: Recombinant Human Active ERK2 Protein , RnD Systems , 1230-KS-010.

Techniques: Recombinant, SYBR Green Assay, Isolation, Reverse Transcription, Colorimetric Assay, Mutagenesis, PCR Cloning, Cloning, Software, Western Blot