Journal: American journal of hypertension

Article Title: Effect of pressure overload on cardioprotection via PI3K-Akt: comparison of postconditioning, insulin, and pressure unloading.

doi: 10.1038/ajh.2010.43

Figure Lengend Snippet: Figure 5 | Effect of IR on phosphorylation status of PDK1 and PTEN. (a) Western blots of phospho-PDK1 and phospho-PTEN for normoxic (control) hearts and (b) phosphoprotein levels for (control) hearts subjected to IR. The data for each protein in each experimental condition are expressed as the percent of phosphoprotein values obtained for hearts that were perfused at the lower pressure. Data represent means ± s.e.m. of 7 hearts/ group/condition. Also shown are representative western blots of each protein and β-actin. *P < 0.05 compared to their counterparts at the lower pressure or to their normoxic counterparts. IR, ischemia reperfusion; PDK1, 3′-phosphoinositide dependent kinase 1; PTEN, phosphatase and tensin homolog on chromosome ten.

Article Snippet: Western blotting was carried out as described previously (i.e., 10% gel, electrophoretic protein transfer to nitrocellulose membrane),24,25 utilizing the following primary antibodies (rabbit; Cell Signaling Technology): Akt, phospho-Akt (Ser473), GSK-3β, phospho-GSK-3β (Ser9), phospho-PDK1 (Ser241), phospho-PTEN (Ser380); goat antirabbit immunoglobin G linked to horse radish peroxidase was used as the secondary antibody; each membrane was reprobed only once for β-actin to confirm equal loading.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

doi: 10.1038/s41598-018-23745-0

Figure Lengend Snippet: Membrane-based antibody macroarray. ( A ) Procedure for membrane-based antibody macroarray. Macroarray membranes were blocked with blocking buffer and then simultaneously reacted with cell lysates and a biotinylated antibody cocktail. After washing, the membranes were incubated with streptavidin-conjugated HRP. Subsequently, multiple proteins were detected using the ECL system. ( B ) Volcano plot of antibody macroarray data created by quantifying the mean spot pixel densities for MA 1 -treated versus non-treated control U937 cell lysate. The x-axis is the fold change between the two samples represented as log 2 ([MA 1 ]/[Control]) and the y-axis is the significance between the two samples represented as −log 10 (p-value). Statistical analyses were performed using the Student’s t test. Green lines show cut-off values (fold change >2, p-value <0.05). The macroarray was conducted with n = 3. ( C ) Macroarray images showing the spots of phospho-RSK1 (Ser380). Upper; non-treated control, lower; MA 1 -treated.

Article Snippet: Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology.

Techniques: Membrane, Blocking Assay, Incubation, Control

Journal: Scientific Reports

Article Title: Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

doi: 10.1038/s41598-018-23745-0

Figure Lengend Snippet: RSK1 is involved in MA 1 -enhanced fibrinolytic activity. ( A , B ) Western blot analysis of phospho-RSK1 (Ser380) and RSK1 in MA 1 -treated U937 cells. MA 1 treatment was performed in the presence of human platelet-poor plasma on a fibrin-coated dish ( A ) or in the absence of plasma on a non-coated dish ( B ) at 37 °C for 1 h. Data were presented as means ± SD (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc test ( ** p < 0.01, versus control). ( C ) Effect of SL0101 on MA 1 -enhanced fibrinolytic activity. For the in vitro fibrin degradation assay, U937 cells with human platelet-poor plasma were added to each well of the 125 I-fibrin-coated 96-well microplate. After incubation for 3 h with MA 1 and SL0101 at the indicated concentrations, 125 I-fibrin degradation products released into the supernatant were quantified using a γ-counter. Data were presented as means ± SD (n = 3). Statistical analyses were performed using two-way ANOVA with Tukey’s post-hoc test ( ** p < 0.01, versus control).

Article Snippet: Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Western Blot, Clinical Proteomics, Control, In Vitro, Degradation Assay, Incubation

Journal: Scientific Reports

Article Title: Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

doi: 10.1038/s41598-018-23745-0

Figure Lengend Snippet: CRISPR/Cas9-mediated RSK1 knockout suppresses MA 1 -enhanced fibrinolytic activity. ( A ) Western blot analysis of RSK1 and RSK2 in CRISPR/Cas9-mediated knockout (KO) U937 cell clones, RSK1#9, RSK1#14, RSK1#17, RSK2#24 and RSK2#27. ( B ) Effect of MA 1 on fibrinolytic activity in CRISPR/Cas9-mediated KO U937 cell clones. CRISPR/Cas9-mediated KO U937 cell clones with human platelet-poor plasma were added to each well of the 125 I-fibrin-coated 96-well microplate. After incubation for 3 h with MA 1 at the indicated concentrations, 125 I-fibrin degradation products released into the supernatant were quantified using a γ-counter. Data were presented as means ± SD (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc test ( * p < 0.05, ** p < 0.01, versus control).

Article Snippet: Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology.

Techniques: CRISPR, Knock-Out, Activity Assay, Western Blot, Clone Assay, Clinical Proteomics, Incubation, Control

Journal: Scientific Reports

Article Title: Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

doi: 10.1038/s41598-018-23745-0

Figure Lengend Snippet: Biologically active MA 1 derivative induces phosphorylation of RSK1. ( A ) Structures of reduced MA 1 , dimethyl MA 1 , 3-LysMA 1 , and 3-BocLysMA 1 . 3-BocLysMA 1 is biologically active, and the other three derivatives are not. ( B ) Western blot analysis of phospho-RSK1 (Ser380) and RSK1 in MA 1 derivative-treated U937 cells. Treatment with 5 µM MA 1 derivatives was performed at 37 °C for 1 h. RedMA 1 ; reduced MA 1 , DMMA 1 ; dimethyl MA 1 . Data were presented as means ± SD (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc test ( ** p < 0.01, versus control).

Article Snippet: Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

doi: 10.1038/s41598-018-23745-0

Figure Lengend Snippet: MA 1 induces the phosphorylation of ERK1/2 and MEK1/2. ( A ) Western blot analysis of phospho-ERK1/2 (Thr202/Tyr204) and ERK1/2, and phospho-MEK1/2 (Ser217/Ser221) and MEK1/2 in MA 1 -treated U937 cells. MA 1 treatment was performed at 37 °C for 1 h. Data were presented as means ± SD (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc test ( * p < 0.05, ** p < 0.01, versus control). ( B ) Western blot time course analysis of phosphorylation of RSK1, ERK1/2, and MEK1/2 in MA 1 -treated U937 cells. U937 cells were treated with 5 μM MA 1 at 37 °C for the indicated times. Data were presented as means ± SD (n = 3). Statistical analyses were performed using two-way ANOVA with Tukey’s post-hoc test ( ** p < 0.01, versus control). ( C ) Effects of PD98059 on MA 1 -induced RSK1 and ERK1/2 phosphorylation. U937 cells were treated with 5 μM MA 1 and 100 µM PD98059 at 37 °C for 1 h. Data were presented as means ± SD (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc test ( ** p < 0.01, versus MA 1 -only treatment).

Article Snippet: Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

doi: 10.1038/s41598-018-23745-0

Figure Lengend Snippet: MA 1 increases the expression of the uPA gene and the secretion of uPA in an RSK1-dependent manner. ( A ) Quantitative real-time PCR for the determination of uPA gene expression levels. U937 cells were treated with MA 1 at the indicated concentration at 37 °C for 1 h. RNA was then extracted and the expression of the uPA gene ( PLAU ) was quantified by qPCR. ( B ) Western blot analysis of uPA in the conditioned medium. U937 cells were treated with MA 1 at the indicated concentration at 37 °C for 3 h. Conditioned media were concentrated ~20-fold and subjected to western blot analysis for the detection of uPA. Data were presented as means ± SD (n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc test ( * p < 0.05, ** p < 0.01, versus control). ( C ) Quantitative real-time PCR for the determination of uPA gene expression levels in CRISPR/Cas9-mediated U937 knockout clones. Knockout clones (RSK1#9, RSK1#14, or RSK2#24) were treated with 5 μM MA 1 at 37 °C for 1 h. RNA was then extracted and the expressions of uPA gene ( PLAU ) were quantified by qPCR. ( D ) Western blot analysis of uPA in the conditioned medium of CRISPR/Cas9-mediated U937 knockout clones. Knockout clones (RSK1#9, RSK1#14, or RSK2#24) were treated with 5 μM MA 1 at 37 °C for 3 h. Conditioned media were concentrated ~20-fold and subjected to western blot analysis for the detection of uPA. Data were presented as means ± SD (n = 3). Statistical analyses were performed using two-way ANOVA with Tukey’s post-hoc test ( * p < 0.05, ** p < 0.01, versus control).

Article Snippet: Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Concentration Assay, Western Blot, Control, CRISPR, Knock-Out, Clone Assay

Journal: Scientific Reports

Article Title: Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

doi: 10.1038/s41598-018-23745-0

Figure Lengend Snippet: A proposed model of MA 1 -mediated MEK-ERK-RSK1 pathway activation and subsequent expression of uPA followed by fibrinolysis enhancement.

Article Snippet: Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Expressing