Journal: bioRxiv

Article Title: EVIDENCE FOR ANGIOTENSIN II AS A NATURALLY EXISTING SUPPRESSOR FOR THE NATRIURETIC PEPTIDE SYSTEM

doi: 10.1101/2023.01.26.525806

Figure Lengend Snippet: (A) Protein expression of different human PKC isoforms in the membrane and cytosol fractions of different HEK293 transfected cell lines (with or without treatment of ANGII). GC-A and NaK-ATPase serve as quality control for the separation of membrane and cytosol fractions. GAPDH serves as loading control. (B) Protein expression of phosphorylated PKC substrates, phosphorylated p38 MAPK, and p38 MAPK in HEK293/GC-A + /AT 1 + cells treated with different PKC modulators. GAPDH serves as loading control. PMA, phorbol 12-myristate 13-acetate. (C) In vitro cGMP generation in HEK293/GC-A + /AT 1 + in response to ANP (10 −8 M) with or without ANGII (10 −8 M) and valsartan (1 μM) or Go6983 (5 μM). Values of cGMP were normalized to vehicle group (blue) under ANP treatment. * indicates P <0.05, one-way ANOVA with Dunnett multiple comparisons test. (D) Hypothetical mechanism underlying the crosstalk between ANGII and GC-A derived from our in vitro studies.

Article Snippet: The following primary antibodies were used: anti-GCA (catalog#MAB48601, R&D System, Minneapolis, MN) at 1:1000 dilution, anti-GCB (catalog#55113-1-AP, Proteintech, Rosemont, IL) at 1:200 dilution, anti-GFP (catalog#TA150041, OriGene, Rockville, MD) at 1:1000 dilution, anti-PKCα (catalog#2056, Cell Signaling Technology, Danvers, MA) at 1:500 dilution, anti-PKCε (catalog#2683, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, anti-Phspho-(Ser) PKC Substrate (catalog#2261, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, anti-NaK-ATPase (catalog#EP1845Y, abcam, Waltham, MA) at 1:2000 dilution, anti-p38 MAPK (catalog#8690, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, anti-Phospho-p38 MAPK (catalog#4511, Cell Signaling Technology, Danvers, MA) at 1:1000 dilution, and anti-GAPDH (catalog#2118, Cell Signaling Technology, Danvers, MA) at 1:5000 dilution.

Techniques: Expressing, Transfection, In Vitro, Derivative Assay

Journal: Biomedicines

Article Title: Inflammatory Cytokine-Induced HIF-1 Activation Promotes Epithelial–Mesenchymal Transition in Endometrial Epithelial Cells

doi: 10.3390/biomedicines11010210

Figure Lengend Snippet: Table of Key Resources.

Article Snippet: Blocking One solution (Nacalai Tesque) was used to block non-specific binding sites for 20 min. Purified primary antibodies were used to probe the membranes, including mouse monoclonal anti-HIF-1α (1:1000; BD Transduction Laboratories, Tokyo, Japan), rabbit polyclonal anti-HIF-2α (1:1000; Novus Biologicals, Centennial, CO, USA), rabbit monoclonal anti-HIF-1 β(1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-p44/42 MAPK (1:2000), rabbit monoclonal anti-p38 MAPK (1:1000), rabbit monoclonal anti-Phospho-p44/42 MAPK (1:1000), rabbit monoclonal anti-Phospho-p38 MAPK (1:1000; Cell Signaling Technology), and mouse monoclonal anti-β-actin (1:5000; Sigma-Aldrich).

Techniques: Blocking Assay, Western Blot, Protease Inhibitor, Purification, Transduction, Recombinant

Journal: Biomedicines

Article Title: Inflammatory Cytokine-Induced HIF-1 Activation Promotes Epithelial–Mesenchymal Transition in Endometrial Epithelial Cells

doi: 10.3390/biomedicines11010210

Figure Lengend Snippet: Involvement of PI3K and MAPK signaling pathways in cytokine-induced HIF-1 activation. ( A ) EM-E6/E7/TERT cells were exposed to TNFα (500 ng/mL) and IL-1β (100 ng/mL) with or without the indicated kinase inhibitors (25 µmol/L LY, 20 µmol/L PD, and 100 µmol/L SC) or 10 mM N-acetyl-L-cysteine (NAC) for 6 h under 20% O 2 conditions, and whole-cell lysates were immunoblotted for HIF-1α and β-actin. LY—LY294002; PD—PD98059; SC—SC-514. ( B , C ) EM-E6/E7/TERT cells were exposed to culture media (–), TNFα (500 ng/mL), and IL-1β (100 ng/mL) under 20% or 5% O 2 conditions for 6 h. After the treatment, whole-cell lysates were immunoblotted for phospho-ERK1/2, ERK1/2 ( B ), phospho-p38, p38 ( C ), and β-actin. ( A – C ) The densitometric analysis’ findings, normalized by -βactin, are displayed as the values above the immunoblots.

Article Snippet: Blocking One solution (Nacalai Tesque) was used to block non-specific binding sites for 20 min. Purified primary antibodies were used to probe the membranes, including mouse monoclonal anti-HIF-1α (1:1000; BD Transduction Laboratories, Tokyo, Japan), rabbit polyclonal anti-HIF-2α (1:1000; Novus Biologicals, Centennial, CO, USA), rabbit monoclonal anti-HIF-1 β(1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-p44/42 MAPK (1:2000), rabbit monoclonal anti-p38 MAPK (1:1000), rabbit monoclonal anti-Phospho-p44/42 MAPK (1:1000), rabbit monoclonal anti-Phospho-p38 MAPK (1:1000; Cell Signaling Technology), and mouse monoclonal anti-β-actin (1:5000; Sigma-Aldrich).

Techniques: Activation Assay, Western Blot

Journal: PLOS ONE

Article Title: Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase

doi: 10.1371/journal.pone.0279011

Figure Lengend Snippet: (A) PRP was stimulated simultaneously by the combination of 0.2 μg/ml of collagen and 10 ng/ml of CXCL12 for the indicated periods. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The levels of GTP-binding Rac in the harvested protein of platelets were determined by the immunoprecipitated Western blot analysis. The representative data are shown. (B, C, D, E) PRP was pretreated for 3 min with 0 μM, 1 μM, 2 μM or 3 μM of NSC23766, and then stimulated simultaneously by the combination of 0.2–0.4 μg/ml of collagen and 10 ng/ml of CXCL12 for 5 min (B, C) or 15 min (D, E). The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. (B) The representative results of platelet aggregation from 3 independent individuals are shown. The black line indicates the percentage of transmittance of each sample (isolated platelets recorded as 0%, and platelet-poor plasma recorded as 100%). The blue line indicates small aggregates (9–25 μm); green line, medium aggregates (25–50 μm); red line, large aggregates (50–70 μm). (C, D, E) The conditioned mixture was centrifuged at 10,000 × g for 2 min at 4°C, and the supernatant was collected. The levels of PDGF-AB (C), sCD40L (D) or phosphorylated-HSP27 (Ser-78) (E) in the supernatant was determined by ELISA. The results from 3 independent individuals are shown. Each value represents the mean ± SEM. *p<0.05, compared to the value of simultaneous stimulation of collagen and CXCL12. (F, G) PRP was pretreated with 3 μM of NSC23766 or vehicle for 3 min, and then stimulated simultaneously by the combination of 0.2–0.4 μg/ml of collagen and 10 ng/ml of CXCL12 for 180 seconds. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. and the levels of phosphorylated p38 MAPK (F) or phosphorylated HSP27 (G) in the lysate of platelets were determined by the Western blot analysis using the antibodies against phospho-p38 MAPK (F), against phospho-HSP27 (G) or against GAPDH. Each histogram shows a quantitative representation of the stimulation with collagen and CXCL12-induced levels obtained from a densitometric analysis. The density levels are expressed as the fold increase compared to the levels of vehicle, presented as lane 1. Each value represents the mean ± SEM of 3 times independent experiments. *p<0.05, compared to the value of vehicle. **p<0.05, compared to the value of simultaneous stimulation of collagen and CXCL12.

Article Snippet: Phospho-specific cofilin antibodies and phospho-specific p38 MAPK antibodies were obtained from Cell Signaling, Inc. (Beverly, MA).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase

doi: 10.1371/journal.pone.0279011

Figure Lengend Snippet: PRP was pretreated with 300 μM of tramadol (A, B, C), 30 μM of fluvoxamine (D, E, F) or vehicle for 3 min, and then simultaneously stimulated by 0.075–0.45 μg/ml of collagen and 10 ng/ml of CXCL12 for 90 seconds (A, D) or 180 seconds (B, C, E, F). The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. (A, D) The protein extracts were harvested as described in Materials and methods, and then GTP-binding Rac was immunoprecipitated using the Rac1 activation assay kit. The immunoprecipitated GTP-binding Rac and pre-immunoprecipitated lysates (Rac) were subjected to Western blot analysis using antibodies against Rac. (B, C, E, F) The levels of phosphorylated p38 MAPK or phosphorylated HSP27 in the cell lysate of platelets were determined by the Western blot analysis using anti-phospho-p38 MAPK antibodies (B, E), anti-phospho-HSP27 (Ser-78) antibodies (C, F) or anti-GAPDH antibodies. Each histogram shows a quantitative representation of the stimulation with collagen and CXCL12-induced levels obtained from a densitometric analysis. The density levels are expressed as the fold increase compared to the levels of vehicle, presented as lane 1. Each value represents the mean ± SEM of 3 times independent experiments. *p<0.05, compared to the value of vehicle. **p<0.05, compared to the value of simultaneous stimulation of collagen and CXCL12.

Article Snippet: Phospho-specific cofilin antibodies and phospho-specific p38 MAPK antibodies were obtained from Cell Signaling, Inc. (Beverly, MA).

Techniques: Binding Assay, Immunoprecipitation, Activation Assay, Western Blot

Journal: PLOS ONE

Article Title: Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase

doi: 10.1371/journal.pone.0279011

Figure Lengend Snippet: PRP was pretreated with 200 μM of morphine (A), 100 nM of reboxetine (B) or vehicle for 3 min, and then simultaneously stimulated by 0.2 μg/ml (A) or 0.4 μg/ml (B) of collagen and 10 ng/ml of CXCL12 for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution, and the levels of phosphorylated p38 MAPK in the cell lysate of platelets were determined by the Western blot analysis using anti-phospho-p38 MAPK antibodies or anti-GAPDH antibodies. The champion data of 3 times independent experiments are shown.

Article Snippet: Phospho-specific cofilin antibodies and phospho-specific p38 MAPK antibodies were obtained from Cell Signaling, Inc. (Beverly, MA).

Techniques: Western Blot

Journal: iScience

Article Title: Drosophila Toll-9 is induced by aging and neurodegeneration to modulate stress signaling and its deficiency exacerbates tau-mediated neurodegeneration

doi: 10.1016/j.isci.2023.105968

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-tau (clone Tau12) (Merck Millipore, Cat#. MAB2241), mouse monoclonal anti-tau-1 (clone PC1C6) (Merk Millipore, Cat#. MAB3420) used for detection of non-phospho tau, mouse monoclonal anti-phospho tau (Ser202/Thr205/Thr208) (clone AT8) (Thermo Fisher Scientific, Cat#. MN1020), rabbit polyclonal anti-phospho tau (Thr217) (Thermo Fisher Scientific, Cat#. 44–744), rabbit polyclonal anti-phospho p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Cat#. 9211S), rabbit monoclonal anti-phospho JNK (Thr183/Tyr185) (Cell Signaling Technology, Cat#. 4668S), rabbit polyclonal anti-phospho Drosophila Akt (Ser505) (Cell Signaling Technology, Cat#. 4054S), mouse monoclonal anti- Drosophila nervana protein (Developmental Studies Hybridoma Bank, Cat#. Nrv5F7) and mouse monoclonal anti-α-tubulin (Sigma-Aldrich, Cat#. T9026) antibodies were purchased.

Techniques: Recombinant, Western Blot, Luciferase, Software

Journal: iScience

Article Title: Drosophila Toll-9 is induced by aging and neurodegeneration to modulate stress signaling and its deficiency exacerbates tau-mediated neurodegeneration

doi: 10.1016/j.isci.2023.105968

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho p38 MAPK (Thr180/Tyr182) antibody , Cell Signaling Technology , Cat# 9211S; RRID: AB_331641.

Techniques: Recombinant, Western Blot, Luciferase, Software