Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of the MAP kinase and NF-κB signaling pathways in mediating ECFP/Ang II-induced NHE3 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that in wild-type mPCT cells, ECFP/Ang II stimulated NHE3 expression significantly, and the response was attenuated by the MEK1/MEK2 kinase inhibitor U0126 and the NF-κB activation inhibitor Ro 106–9920, respectively. However, the MEK inhibitor PD 980659 and the p38 MAP kinase inhibitor SB202196 failed to attenuate the effect of ECFP/Ang II on NHE3 expression. Panel ( B ) shows that in Agtr1a -/- mPCT cells, ECFP/Ang II failed to stimulate NHE3 expression, and the inhibitors of the MAP kinases and NF-κB signaling pathways had no significant effects on NHE3 expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced Na + /HCO 3 - cotransporter expression in wild-type mPCT cells. Panel ( A ) shows that ECFP/Ang II significantly increased Na + /HCO 3 - expression, and the response was attenuated by losartan but not by PD123319, suggesting a dominant role of AT 1 receptors in mPCT cells. Panel ( B ) shows that the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659 attenuated the effects of ECFP/Ang II on expression, but the p38 MAP kinase inhibitor SB202196 had no effect on Na + /HCO 3 - expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that ECFP/Ang II increased NF-κB, p65 expression in wild-type mPCT cells, and the response was attenuated by both losartan and PD123319, supporting an important role of AT 1 and AT 2 receptors in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. Panel ( B ) shows that ECFP/Ang II alone had no significant effect on NF-κB, p65 expression in Agtr1a -/- mPCT cells, but both losartan and PD123319 potentiated this response. Panel ( C ) shows that in wild-type mPCT cells, the effect of ECFP/Ang II on NF-κB, p65 expression was attenuated by the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659, respectively. However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. ** p < 0.01 vs. control WT or Agtr1a -/- mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II, or Agtr1a -/- mPCT cells transfected with ECFP/ANG II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Journal of Biological Chemistry

Article Title: Role of Sphingosine-1-phosphate Phosphatase 1 in Epidermal Growth Factor-induced Chemotaxis

doi: 10.1074/jbc.m404907200

Figure Lengend Snippet: FIG. 6. SPP-1 overexpression abro- gates EGF-induced ERK 1/2 activation but not EGFR tyrosine phosphoryla- tion. HEK 293 cells stably expressing vec- tor or SPP-1 were serum-starved for 16 h and treated without or with EGF (100 ng/ ml) for 5 min. A, cells were lysed, and equal amounts of proteins were immunoprecipi- tated with anti-EGFR and analyzed by Western blotting with anti-phosphoty- rosine antibody (upper panel). Blots were then stripped and reprobed with anti- EGFR (bottom panel) as a loading control. B, equal amounts of total lysates were also analyzed by immunoblotting with an- ti-phosphotyrosine; activation of ERK (C) and p38 (D) was determined with phos- pho-specific anti-ERK1/2 and p38 anti- bodies, respectively. Blots were stripped and reprobed with ERK2 or p38 antibod- ies to demonstrate equal loading. Num- bers indicate relative intensities deter- mined by densitometry of scanned blots.

Article Snippet: AntiEGF receptor polyclonal antibody and phospho-p38 and ERK2 antibodies (Santa Cruz), phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY), p38, and phospho-ERK1/2 antibodies (Cell Signaling Technology, Beverly, MA) were used as primary antibodies.

Techniques: Over Expression, Activation Assay, Stable Transfection, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p < 0.05 compared to the negative control group.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Inhibition, Western Blot, Phospho-proteomics, In Vitro, Migration, Negative Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon up-regulates p38β expression in hepatic cells. Pokemon was delivered by expression plasmid pcDNA3.1(−)-Pokemon with pcDNA3.1(−) as a negative control. Pokemon silencing was triggered by si-RNA. ( a ) Targeted expression or silencing of Pokemon in HepG2 cells. Cells were collected at 60 h after transfection or silencing (Left panel). And the quantification of western blot data was displayed on the Right panel; ( b ) Real-time quantitative polymerase chain reaction (qPCR) at 48 h after transfection in HepG2 cells. Upper panel: Ectopic expression of Pokemon; lower panel: Silencing of Pokemon; ( c ) Ectopic expression of Pokemon in HL7702 cells. Upper panel: Western blot in which cells were lysed and total proteins were collected at 36, 48, 60 and 72 h, respectively. Lower panel: Real-time qPCR at 48 and 60 h after transfection; ( d ) Silencing of Pokemon in BEL7404 cells. Upper panel: Western blot; lower panel: Real-time qPCR. * p < 0.05 compared to the negative control. N.C. means negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Negative Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

doi: 10.3390/ijms140713511

Figure Lengend Snippet: Pokemon stimulates p38β promoter activity. ( a ) ChIP assays in HepG2 and BEL7404 cells. Protein-DNA complexes are immunoprecipitated either with anti-Pokemon antibody or anti-IgG as negative control, followed by PCR with primers specific to p38β promoter sequence and agarose-gel electrophoresis for visualization. Total lysates were used as the input samples and positive control; ( b ) Dual luciferase reporter assay. Luciferase activities were normalized to Renilla activity. Y axis stands for the relative fold changes of activity as the pcDNA3.1(−)-Pokemon plasmid increases. * p < 0.05 compared to the negative control.

Article Snippet: Antibodies used are as follows: Pokemon antibody (Sigma, St. Louis, MO, USA), p38α MAPK (7D6) Rabbit mAb (CST, Danvers, MA, USA), p38β MAPK (c28c2) Rabbit mAb (CST), Phospho-p38 MAPK Pathway Sampler Kit (CST) and Actin antibody (Beyotime, Shanghai, China).

Techniques: Activity Assay, Immunoprecipitation, Negative Control, Sequencing, Agarose Gel Electrophoresis, Positive Control, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Hibiscus sabdariffa L.: A potent natural neuroprotective agent for the prevention of streptozotocin-induced Alzheimer's disease in mice.

doi: 10.1016/j.biopha.2020.110303

Figure Lengend Snippet: Fig. 6. Effects of Hibiscus extracts and celecoxib on the activity of p38MAPK (A), and COX-2 (B), and on concentration of mPGES-1 (C) and PGE2 (D). p38MAPK and COX-2 levels in celecoxib (30 mg/kg), white and red Hibiscus (200 mg/kg each) groups were decreased compared to STZ (3 mg/kg, ICV) group and increased compared to the normal group. mPGES-1 and PGE2levels were normalized in the three treated groups. Panel 4 represents immunoblot analysis shown in Fig. 6.A. Panel 5 represents immunoblot analysis shown in Fig. 6.B. Data are expressed as means ± S.D. The significance of the dif- ference between means was tested by ANOVA followed by Tukey Kramer multiple compar- isons test. * P < 0.05 vs normal; @ P < 0.05 vs STZ-treated group; # P < 0.05 vs celecoxib. n = 6 mice, DF = 29. For p38MAPK: F = 354.1, R2 = 0.9827. For COX-2: F = 350.5, R2 = 0.9825. For m-PGES-1: F = 430.4, R2 = 0.9857. For PGE2: F = 354.3, R2 = 0.9827.

Article Snippet: The antibodies used were rabbit BACE1 monoclonal [EPR19523] (abcam, ab183612), mouse Cox-2 (H-3) monoclonal (Santa cruze Inc, sc-376861), rabbit PSENEN polyclonal (Boster Biological Technology, A04504) and rabbit p38 MAPK polyclonal (Biorbyt Ltd., orb127559).

Techniques: Activity Assay, Concentration Assay, Western Blot

Journal: International journal of molecular medicine

Article Title: Rhododendron album Blume inhibits iNOS and COX-2 expression in LPS-stimulated RAW264.7 cells through the downregulation of NF-κB signaling.

doi: 10.3892/ijmm.2015.2107

Figure Lengend Snippet: Figure 4. Rhododendron album Blume methanol extract (RAME) suppresses the phosphorylation of mitogen-activated protein kinase (MAPK)‑associated molecules in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cells were pre-incubated with RAME for 12 h and then incubated with LPS (0.5 µg/ml) for 20 min. The total protein lysate was subjected to western blot analysis. The ratio of the immunointensity of p-ERK1/2, p-JNK and p-p38 was calculated. Total ERK1/2, JNK and p38 (ERK1/2, JNK and p38) were used as a control for the amount of protein in the same samples. A representative blot of each experiment is shown with the densitometric analysis corresponding to the means ± SEM of 3 independent experiments.

Article Snippet: Each membrane was then incubated for 1 h in 5% skim milk in TBS-T buffer (0.1 M Tris-HCl, pH 7.4, 0.9% NaCl, 0.1% Tween-20) to block non-specific binding and was then incubated with primary antibodies that recognized iNOS (Cat. no. ADI-905-431, 1:1,000; obtained from Enzo Life Sciences, Farmingdale, NY, USA), COX-2 (Cat. no. sc-1747, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (Cat. no. #4967, 1:2,000; Cell Signaling Technology, Danvers, MA, USA), PARP (Cat. no. #9542; Cell Signaling Technology), the total forms of extracellular signal-regulated kinase (ERK)2 (sc-154), p38 MAPK (sc-7149), c-Jun N-terminal kinase (JNK)1/3 (sc-474, 1:1,000; Santa Cruz Biotechnology), the phosphorylated forms of p38 MAPK (ADI-KAP-MA022) and JNK1/2 (KAP-SA011, 1:1,000; Enzo Life Sciences, Inc.) and the phosphorylated forms of ERK (#9106, 1:1,000; Cell Signaling Technology).

Techniques: Phospho-proteomics, Incubation, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8 + T Cell Receptor Signaling

doi: 10.3389/fimmu.2017.00265

Figure Lengend Snippet: Distal T cell receptor-mediated signaling is compromised in maternal antibiotic treatment (MAT) naïve CD8 + T cells . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were analyzed for (A) expression of Erk2 (CTRL, n = 5; MAT, n = 4), (B) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 5; MAT, n = 4), (C) expression of pErk1/2 (CTRL, n = 5; MAT, n = 4), and (D) expression of c-Rel (CTRL, n = 6; MAT, n = 3) gating on naïve (CD44 − ) CD8 + T cells by flow cytometry. To analyze pErk1/2 expression (B,C) , T cells were left unstimulated (−) or stimulated (+) with anti-CD3/anti-CD28 or phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2 min at 37°C. Representative graphs (CTRL, white; MAT, black; dashed line, negative staining control) and analysis of the median fluorescence intensity (MFI) or percentage are shown. Data are representative of two independent experiments, presented as mean + SEM and were analyzed by one-way ANOVA with Holm–Sidak posttest (B,C) or unpaired two-tailed Student’s t -test (D) . * p < 0.05, ** p < 0.01.

Article Snippet: Single cell suspensions of lymphocytes isolated from the spleen, MLN, or PEC of uninfected and infected infant mice were stained with optimal concentrations of the following antibodies and reagents: CD3ε (clone 145-2C11), CD8α (clone 53-6.7), CD25 (clone PC61), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (Clone H1.2F3), TNF-α (clone MP6-XT22), IFN-γ (clone XMG1.2), pTyr (clone pY20), ZAP-70 (clone 1E7.2), phosphorylated ZAP-70 (pZAP-70) Tyr319 (clone n3kobu5; eBioscience), Erk2 (clone REA186; Miltenyi Biotec), phospho-Erk-1/2 (pErk1/2) Thr202/Tyr 204 (clone 4B11B69), c-Rel (clone REA397; Miltenyi Biotec), Ki-67 (clone B56; BD Biosciences), annexin V, and 7-aminoactinomycin D (7-AAD).

Techniques: Control, Expressing, Flow Cytometry, Negative Staining, Fluorescence, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8 + T Cell Receptor Signaling

doi: 10.3389/fimmu.2017.00265

Figure Lengend Snippet: Maternal antibiotic treatment (MAT) effector CD8 + T cells do not sustain phosphorylation of Erk-1/2 . Total T cells enriched from the spleens of day of life 15 control (CTRL) and MAT infant mice were stimulated with anti-CD3/anti-CD28 for 24, 48, and 72 h and analyzed for (A) percentage of Erk2 + cells (CTRL, n = 7; MAT, n = 6), (B) expression of Erk2 (CTRL, n = 7; MAT, n = 6), (C) percentage of phospho-Erk-1/2 (pErk1/2) positive cells (CTRL, n = 7; MAT, n = 6), and (D) expression of pErk1/2 gating on effector (CD44 + ) CD8 + T cells (Teff). (E) Percentage of CD8 + Teff cells expressing low or high levels of Erk2 (Erk2 lo and Erk2 hi , respectively) and gating analysis of their corresponding pErk1/2-expressing populations at 48 h post-stimulation. Data are representative of two independent experiments and presented as mean + SEM. * p < 0.05, **** p < 0.0001, one-way ANOVA with Holm–Sidak posttest.

Article Snippet: Single cell suspensions of lymphocytes isolated from the spleen, MLN, or PEC of uninfected and infected infant mice were stained with optimal concentrations of the following antibodies and reagents: CD3ε (clone 145-2C11), CD8α (clone 53-6.7), CD25 (clone PC61), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (Clone H1.2F3), TNF-α (clone MP6-XT22), IFN-γ (clone XMG1.2), pTyr (clone pY20), ZAP-70 (clone 1E7.2), phosphorylated ZAP-70 (pZAP-70) Tyr319 (clone n3kobu5; eBioscience), Erk2 (clone REA186; Miltenyi Biotec), phospho-Erk-1/2 (pErk1/2) Thr202/Tyr 204 (clone 4B11B69), c-Rel (clone REA397; Miltenyi Biotec), Ki-67 (clone B56; BD Biosciences), annexin V, and 7-aminoactinomycin D (7-AAD).

Techniques: Phospho-proteomics, Control, Expressing

Journal: Frontiers in Immunology

Article Title: Protective Effects of Lactic Acid Bacteria Against TLR4 Induced Inflammatory Response in Hepatoma HepG2 Cells Through Modulation of Toll-Like Receptor Negative Regulators of Mitogen-Activated Protein Kinase and NF-κB Signaling

doi: 10.3389/fimmu.2018.01537

Figure Lengend Snippet: Total cellular fluid (TCF) of lactic acid bacteria (LABs) downregulated expression of p38 mitogen-activated protein kinase (MAPK) in hepatoma HepG2 cells. Confluence HepG2 cells were pre-stimulated with LABs TCF (50 µl/ml) for 48 h, then stimulated with LPS for different time intervals (0, 30, 60, 90, and 120 min). Western blot was performed to determine the phosphorylation of p38 MAPK at the indicated time points. The bar graphs represent the results of three independent experiments. The image J software was used to determine the intensities of proteins bands. Different superscript letters indicate significant differences at the 0.05 level.

Article Snippet: The phosphorylation of p38 and degradation of p65 were evaluated by incubating membranes with MAPK Phospho-p38α (T180/Y182) antibody (p-p38α, Cat. #MAB8691, R&D Systems, MN, USA); NF-κB phospho-p65 (p-p65, Cat. #9242); and β-actin antibody (Cat. #4970) from Cell Signaling Technology (Beverly, MA, USA) overnight at RT.

Techniques: Bacteria, Expressing, Western Blot, Phospho-proteomics, Software