Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: (A) Representative cPLA2 and GAPDH Western blots in iliac arteries from early- (n = 3) versus advanced-stage (n = 3) CVD patients. (B) Scatter dot plots (arithmetic mean ± SEM) of cPLA2 protein expression normalized to GAPDH in iliac arteries from early- (n = 14) versus advanced-stage (n = 23) CVD patients. *P < 0.05 vs. early-stage CVD group (unpaired Mann-Whitney test).

Article Snippet: EFEMP1, GUCY1B2, KDM5D, cPLA2, and SHMT1 were detected with a rabbit anti-human EFEMP1 antibody (1:1000, Abcam), rabbit anti-human GUCY1B2 antibody (1:500, Thermo Fisher Scientific), rabbit anti-human KDM5D (Jarid 1D, 1:5000, Novus Biologicals), rabbit anti-human PLA2G4A antibody (1:500, ProSci Incorporated), and rabbit anti-human SHMT1 (1 250, MilliporeSigma), respectively, followed by a goat anti-rabbit IR dye 680 secondary antibody (1:7500, LI-COR Biosciences).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: (A) Scatter dot plots (arithmetic mean ± SEM) (n = 5–6, μg/mg protein) of calcium content in aortic tissue of mice receiving vehicle (CTR) or high-dosed cholecalciferol (vD), without or with additional AACOCF3 (AAC) treatment. (B) Scatter dot plots (arithmetic mean ± SEM) (n = 5–6; arbitrary units [a.u.]) of Pla2g4a relative mRNA expression in aortic tissue of mice receiving vehicle or high-dosed cholecalciferol, without or with additional AACOCF3 treatment. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice; †P < 0.05 vs. vD-treated mice (1-way ANOVA with Tukey-HSD post-hoc test).

Article Snippet: EFEMP1, GUCY1B2, KDM5D, cPLA2, and SHMT1 were detected with a rabbit anti-human EFEMP1 antibody (1:1000, Abcam), rabbit anti-human GUCY1B2 antibody (1:500, Thermo Fisher Scientific), rabbit anti-human KDM5D (Jarid 1D, 1:5000, Novus Biologicals), rabbit anti-human PLA2G4A antibody (1:500, ProSci Incorporated), and rabbit anti-human SHMT1 (1 250, MilliporeSigma), respectively, followed by a goat anti-rabbit IR dye 680 secondary antibody (1:7500, LI-COR Biosciences).

Techniques: Expressing, Control

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: Scatter dot plots (arithmetic mean ± SEM) (n = 6, arbitrary units [a.u.]) of PLA2G4A (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs following silencing with negative control siRNA (Neg.si) or cPLA2 siRNA (cPLA2si), without or with additional treatment with β-glycerophosphate (Pi). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Neg.si-silenced HAoSMCs; †P < 0.05, †††P < 0.001 vs. Neg.si-silenced and Pi-treated HAoSMCs (1-way ANOVA with Games-Howell post-hoc test for A or with Tukey-HSD post-hoc test for C and Steel-Dwass method for B and D).

Article Snippet: EFEMP1, GUCY1B2, KDM5D, cPLA2, and SHMT1 were detected with a rabbit anti-human EFEMP1 antibody (1:1000, Abcam), rabbit anti-human GUCY1B2 antibody (1:500, Thermo Fisher Scientific), rabbit anti-human KDM5D (Jarid 1D, 1:5000, Novus Biologicals), rabbit anti-human PLA2G4A antibody (1:500, ProSci Incorporated), and rabbit anti-human SHMT1 (1 250, MilliporeSigma), respectively, followed by a goat anti-rabbit IR dye 680 secondary antibody (1:7500, LI-COR Biosciences).

Techniques: Expressing, Negative Control

Journal: Cancers

Article Title: Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival

doi: 10.3390/cancers13184691

Figure Lengend Snippet: Ac-DNMT1 and TIP60 protein levels positive correlate in metastatic melanoma. ( A ) Comparison of TIP60 mRNA expression using TCGA SKCM dataset. ( B ) Representative IHC images of TIP60 in the TMA melanoma cohort. Scale bars = 20 μm. ( C , D ) Correlation between TIP60 and ac-DNMT1 protein levels in stage III ( C ) and stage IV ( D ) metastatic FFPE tissues. ( E ) Comparison of TIP60 H-scores in stage IV patients with high and low ac-DNMT1 protein levels. The best-fit line (straight line) and the 95% CI (dotted line) are shown in grey. Data represent the mean ± SD. ns : not significant, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: IHC staining was performed as previously described [ , ], using anti-human DNMT1 rabbit polyclonal antibody (Ab, 1:100 dilution, Cat# NB100-264, Novus Biologicals, Centennial, CO, USA), anti-human ac-DNMT1 rabbit polyclonal Ab (1:100 dilution, Cat# A5595, ABclonal, Woburn, MA, USA), anti-human Ki-67 mouse monoclonal Ab (1:100 dilution, Cat# M7240, DAKO, Santa Clara, CA, USA), anti-human TIP60 rabbit polyclonal Ab (1:100 dilution, Cat# 10827-1-AP, Proteintech, Rosemont, IL, USA), and anti-human USP7 rabbit monoclonal Ab (1:100 dilution, Cat# A700-072, Bethyl, Montgomery, TX, USA) for the TMA slide.

Techniques: Comparison, Expressing

Journal: Cancers

Article Title: Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival

doi: 10.3390/cancers13184691

Figure Lengend Snippet: Ac-DNMT1 and USP7 protein levels positive correlate in metastatic melanoma. ( A ) Representative IHC images of USP7 in the TMA melanoma cohort. Scale bars = 20 μm. ( B , C ) Correlation between USP7 and ac-DNMT1 protein levels in stage III ( B ) or stage IV ( C ) metastatic FFPE tissues. ( D ) Comparison of USP7 H-scores in stage IV patients with high or low ac-DNMT1 protein levels. ( E ) Comparison of USP7 H-scores in normal organ tissues, stage III, and stage IV metastasis from the TMA melanoma cohort. ( F , G ) Correlation between USP7 and TIP60 protein levels in stage III ( F ) or stage IV ( G ) metastatic FFPE tissues. The best-fit line (straight line) and the 95% CI (dotted line) are shown in grey. Data represent the mean ± SD. ns : not significant, * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Article Snippet: IHC staining was performed as previously described [ , ], using anti-human DNMT1 rabbit polyclonal antibody (Ab, 1:100 dilution, Cat# NB100-264, Novus Biologicals, Centennial, CO, USA), anti-human ac-DNMT1 rabbit polyclonal Ab (1:100 dilution, Cat# A5595, ABclonal, Woburn, MA, USA), anti-human Ki-67 mouse monoclonal Ab (1:100 dilution, Cat# M7240, DAKO, Santa Clara, CA, USA), anti-human TIP60 rabbit polyclonal Ab (1:100 dilution, Cat# 10827-1-AP, Proteintech, Rosemont, IL, USA), and anti-human USP7 rabbit monoclonal Ab (1:100 dilution, Cat# A700-072, Bethyl, Montgomery, TX, USA) for the TMA slide.

Techniques: Comparison

Journal: Cancers

Article Title: Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival

doi: 10.3390/cancers13184691

Figure Lengend Snippet: Low ac-DNMT1 protein level is a predictive factor for poor MSS. ( A ) MSS curves for stage IV metastasis from the TMA cohort. Patients were divided according to the mean IHC H-scores of ac-DNMT1. ( B ) MSS curves for stage IV metastasis from the TMA cohort. Patients were divided according to the mean IHC H-scores of ac-DNMT1 together with DNMT1 ( B ), TIP60 ( C ), USP7 ( D ), and Ki-67 ( E ) into high and low groups, respectively.

Article Snippet: IHC staining was performed as previously described [ , ], using anti-human DNMT1 rabbit polyclonal antibody (Ab, 1:100 dilution, Cat# NB100-264, Novus Biologicals, Centennial, CO, USA), anti-human ac-DNMT1 rabbit polyclonal Ab (1:100 dilution, Cat# A5595, ABclonal, Woburn, MA, USA), anti-human Ki-67 mouse monoclonal Ab (1:100 dilution, Cat# M7240, DAKO, Santa Clara, CA, USA), anti-human TIP60 rabbit polyclonal Ab (1:100 dilution, Cat# 10827-1-AP, Proteintech, Rosemont, IL, USA), and anti-human USP7 rabbit monoclonal Ab (1:100 dilution, Cat# A700-072, Bethyl, Montgomery, TX, USA) for the TMA slide.

Techniques:

Journal: Cancers

Article Title: Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival

doi: 10.3390/cancers13184691

Figure Lengend Snippet: Multivariate analyses including TIP60 protein levels for 4-year MSS 1 in stage IV melanoma patients.

Article Snippet: IHC staining was performed as previously described [ , ], using anti-human DNMT1 rabbit polyclonal antibody (Ab, 1:100 dilution, Cat# NB100-264, Novus Biologicals, Centennial, CO, USA), anti-human ac-DNMT1 rabbit polyclonal Ab (1:100 dilution, Cat# A5595, ABclonal, Woburn, MA, USA), anti-human Ki-67 mouse monoclonal Ab (1:100 dilution, Cat# M7240, DAKO, Santa Clara, CA, USA), anti-human TIP60 rabbit polyclonal Ab (1:100 dilution, Cat# 10827-1-AP, Proteintech, Rosemont, IL, USA), and anti-human USP7 rabbit monoclonal Ab (1:100 dilution, Cat# A700-072, Bethyl, Montgomery, TX, USA) for the TMA slide.

Techniques:

Journal: Antioxidants

Article Title: PCSK9 Regulates Nox2-Mediated Platelet Activation via CD36 Receptor in Patients with Atrial Fibrillation

doi: 10.3390/antiox9040296

Figure Lengend Snippet: Intra-signalling pathway of platelet activation PCSK9-mediated: p38MAP Kinase, p47 phox and PLA 2 phosphorylation. p38 phosphorylation, p47 phox phosphorylation and cPLA2 phosphorylation was analysed in platelets incubated with PCSK9 (1–2 ng/mL) and stimulated with subthreshold concentration (STC) of collagen (0.25 µg/mL) ( A–C ) in presence or less of anti-CD36, anti-LOX1, anti-PCSK9 ( A–C ) and incubated with or without SB ( A ), Nox2ds-tat ( B ) and AACOCF3 ( C ) ( n = 3 independent experiments; * p < 0.05 ** p < 0.001 in quantitative analysis). A representative Western blot of p38, p47phox and cPLA2 phosphorylation ( B–C ).

Article Snippet: Moreover, membranes were incubated overnight at 4 °C with rabbit polyclonal anti-p-p47phox (1:1000; ab-795, Abcam, Cambridge, UK), anti-p-p38 and anti-p-cPLA2 antibody (1:1000; sc-7973 and sc34392, Santa Cruz Biotechnology, Dallas, TX, USA; respectively), and anti-p47phox, anti-p38 and anti-cPLA2 antibody (1:1000; sc-17845, sc-7973 and sc-376618, Santa Cruz Biotechnology, TX, USA; respectively).

Techniques: Activation Assay, Phospho-proteomics, Incubation, Concentration Assay, Western Blot

Journal: Mediators of Inflammation

Article Title: Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines

doi: 10.1155/2016/9160540

Figure Lengend Snippet: Cross-linking GD1b derived gangliosides by mAbAA4 increased cPLA 2 phosphorylation and induced COX-2 expression. Either RBL-2H3 mast cells were stimulated via Fc ε RI, by sensitizing the cells with IgE anti-TNP and stimulating with DNP 48 -HSA (50 ng/mL), or cells were incubated with mAbAA4 (1, 2.5, 5, and 10 μ g/mL) for 5 min (cPLA 2 phosphorylation) or for 1 h and then rinsed and cultured for an additional 3 h (COX-2 expression). Total cell lysates were immunoblotted with antibodies against p-cPLA 2 , cPLA 2 , COX-2, and α / β -tubulin (housekeeping protein) and the mean optical density of the bands was determined. Densitometry of the changes in expression and phosphorylation of proteins were corrected for α / β -tubulin. Data were expressed as the fold of nonstimulated (NS) cells. (a) Ratio of phosphorylated cPLA 2 /total cPLA 2 ; (b) a representative blot from (a); (c) ratio of COX-2/ α / β -tubulin; (d) a representative blot from (c). Data is expressed as the mean ± SD of three independent experiments. ∗ P < 0.05 between experimental samples and the nonstimulated (NS) cells. # P < 0.05 between experimental samples and Fc ε RI stimulated cells.

Article Snippet: Rabbit polyclonal antibody anti-human phospho-cPLA 2 ; rabbit polyclonal antibody anti-human cPLA 2 ; rabbit mAb anti-human phospho-ERK1/2; rabbit mAb anti-rat ERK1/2; rabbit mAb anti-human phospho-JNK1/2; rabbit polyclonal antibody anti-human JNK1/2; rabbit mAb anti-human phospho-p38; rabbit polyclonal antibody anti-human anti-p38, and rabbit polyclonal antibody anti-human α / β -tubulin were purchased from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Derivative Assay, Phospho-proteomics, Expressing, Incubation, Cell Culture

Journal: Mediators of Inflammation

Article Title: Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines

doi: 10.1155/2016/9160540

Figure Lengend Snippet: Cross-linking GD1b derived gangliosides with mAbAA4 induced MAP kinase phosphorylation in mast cells. RBL-2H3 cells were either stimulated via Fc ε RI, where cells were sensitized with IgE anti-TNP and stimulated with DNP 48 -HSA (50 ng/mL) or incubated with mAbAA4 (1, 2.5, 5, and 10 μ g/mL) for 10 min. Total cell lysates were immunoblotted with antibodies against phospho-ERK1/2 (p-ERK1/2), ERK1/2, phospho-JNK1/2 (p-JNK1/2), JNK1/2, phospho-p38 (p-p38), p38, and α / β -tubulin (housekeeping protein) and the mean optical density of the bands was determined. Densitometry of the changes in expression and phosphorylation of proteins were corrected for α / β -tubulin. Data is expressed as the fold of nonstimulated (NS) cells. (a) Ratio of phosphorylated ERK1/2/total ERK1/2; (b) a representative blot from (a); (c) ratio of phosphorylated JNK1/2/total JNK1/2; (d) a representative blot from (c); (e) ratio of phosphorylated p38/total p38; (f) a representative blot from (e). Data is expressed as the mean ± SD of three independent experiments. ∗ P < 0.05 between experimental samples and the nonstimulated (NS) cells. # P < 0.05 between experimental samples and Fc ε RI stimulated cells.

Article Snippet: Rabbit polyclonal antibody anti-human phospho-cPLA 2 ; rabbit polyclonal antibody anti-human cPLA 2 ; rabbit mAb anti-human phospho-ERK1/2; rabbit mAb anti-rat ERK1/2; rabbit mAb anti-human phospho-JNK1/2; rabbit polyclonal antibody anti-human JNK1/2; rabbit mAb anti-human phospho-p38; rabbit polyclonal antibody anti-human anti-p38, and rabbit polyclonal antibody anti-human α / β -tubulin were purchased from Cell Signaling Technology Inc. (Danvers, MA).

Techniques: Derivative Assay, Phospho-proteomics, Incubation, Expressing