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: 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: 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

Journal: Experimental eye research

Article Title: TGF-β1-induced apoptosis in retinal endothelial cells is implicated in retinal vein occlusion.

doi: 10.1016/j.exer.2024.110168

Figure Lengend Snippet: Fig. 4. TGF-β1 signaling activation induces apoptosis in HRMECs by activating p38/JNK signaling A. ELISA analysis of extracellular TGF-β1 and phospho-Smad2/3 levels in HRMECs treated with recombinant TGF-β1, OGD/R, or OGD/R + SB431542 (TGF-β in hibitor). B. Flow cytometry analysis of apoptosis in HRMECs treated as in (A). C. Immunoblotting analysis of phospho-p38 and phospho-JNK levels in HRMECs treated as in (A). D. ROS measurement in HRMECs treated as in (A). N = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Membranes were then incubated with primary antibodies targeting phospho-p38 (Abcam, ab4822), total p38 (R&D Systems, MAB8691), phospho-JNK (Proteintech, 80024-1-RR), total JNK (Novus Biologicals, NBP1-92077), and GAPDH (Sigma-Aldrich, G8795) at 4 ◦C for 16 h. After washing, membranes were exposed to species-appropriate HRP-linked secondary antibodies (Rockland Immunochemicals, 610- 103-122 and 611-103-122).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Flow Cytometry, Western Blot

Journal: Experimental eye research

Article Title: TGF-β1-induced apoptosis in retinal endothelial cells is implicated in retinal vein occlusion.

doi: 10.1016/j.exer.2024.110168

Figure Lengend Snippet: Fig. 6. Effects of TGF-β1 inhibition on UV-induced changes in HRMECs A. ELISA analysis of extracellular TGF-β1 and phospho-Smad2/3 levels in HRMECs treated with UV or UV + SB431542. B. Flow cytometry analysis of apoptosis in HRMECs treated as in (A). C. Immunoblotting analysis of phospho-p38 and phospho-JNK levels in HRMECs treated as in (A). D. ROS measurement in HRMECs treated as in (A). N = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Membranes were then incubated with primary antibodies targeting phospho-p38 (Abcam, ab4822), total p38 (R&D Systems, MAB8691), phospho-JNK (Proteintech, 80024-1-RR), total JNK (Novus Biologicals, NBP1-92077), and GAPDH (Sigma-Aldrich, G8795) at 4 ◦C for 16 h. After washing, membranes were exposed to species-appropriate HRP-linked secondary antibodies (Rockland Immunochemicals, 610- 103-122 and 611-103-122).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: (a) TGFβ stimulates p38α activity, inhibited by SB2036580 or β-LGND. Bar graph is the mean±SD from 3 exps combined. *p < 0.05 vs. control, +p < 0.05 for TGFβ vs same + SB2036580 or β-LGND. (b) TGFβ inhibits KLF15 mRNA and protein in cardiomyocytes, blocked by the p38 antagonist SB2036580 (0.1μM) (c) TAK1 activating phosphorylation is stimulated by AngII or TGFβ, inhibited by β-LGND. *p<0.05 vs. control, + p<0.05 for TGFβ or AngII vs same plus β-LGND, n=3 exps. (d) TAK1 siRNA diminishes TGFβ or AngII-stimulated p38α activity. The latter was seen as phosphorylation at tyrosine182. *p<0.05 vs control, +p<0.05 for TGFβ or AngII vs same + β-LGND, n=3 exps. TAK1 siRNA validation is also shown. (e) Flow cytometry analysis of β-LGND inhibition of phospho-kinases due to cAMP/PKA. *p<0.05 for control vs. AngII-stimulated phospho-TAK1, phospho-p38α, or KLF15 proteins. +p<0.05 for AngII vs AngII + β-LGND, ++p<0.05 for AngII + β-LGND vs same + either H-89 (PKA inhibitor) or RP-8-Br-cAMP (cAMP inhibitor), n=3 exps.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Activity Assay, Control, Phospho-proteomics, Biomarker Discovery, Flow Cytometry, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Estrogen receptor beta maintains expression of KLF15 to prevent cardiac myocyte hypertrophy in female rodents

doi: 10.1016/j.mce.2017.11.004

Figure Lengend Snippet: AngII acting through TGFβ stimulates a TAK1-p38α kinase axis that inhibits KLF15 expression and nuclear localization of the protein. This contributes to increased gene expression and cardiomyocyte hypertrophy. ERβ acting through protein kinase A opposes TAK1-p38α activation. This restores KLF15 abundance and nuclear localization, contributing in part to inhibition of AngII-induced gene expression and cardiomyocyte hypertrophy.

Article Snippet: Additional antibodies and phospho-specific antibodies used for immuno-blots were obtained from the followings: Cell Signaling Technology (Danvers, MA) TAK1 (D94D7) (#5206), Phospho-ATF-2 (Thr71) (#9221), Phospho-TAK1 (Thr187) (#4536); Santa Cruz, Biotechnology (Dallas, TX), KLF15 (A5) (SC-271675), GAPDH (0411) (sc-47724), MYH7 (A4.951) (sc-53090), Actin (2Q1055) (sc-58673), p38 Antibody (A-20) (sc-535), phospho-p38 (Thr 180/Tyr 182) (sc-17852-R); (Boster Biological Technology, Pleasanton, CA), ACTA2 (M01072–1).

Techniques: Expressing, Gene Expression, Activation Assay, Inhibition

Journal: Journal of Biological Chemistry

Article Title: JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

doi: 10.1074/jbc.m109.061515

Figure Lengend Snippet: FIGURE 3. FBS induces the phosphorylation of JNK, p38, and ERK in CHO-K1cells.Serum-starvedCHO-K1cellsweretreatedwith7.5%FBSforthe indicated times. Total and phosphorylated JNK, p38, and ERK were monitored by Western blot.

Article Snippet: Rabbit anti-cPLA2 , anti-phospho-Ser-505-cPLA2 , anti-JNK, anti-phospho-Thr-183/Tyr-185-JNK, anti-p38, antiphospho-Thr-180/Tyr-182-p38, anti-p44/42, and anti-phosphoThr-202/Tyr-204-p44/p42 antibodies were from Cell Signaling; chicken anti-ADRP was from GenWay Biotech; rabbit anti-glyceraldehyde-3-phosphate dehydrogenase was from Ambion, and rabbit anti-CERK fromAbcam.

Techniques: Phospho-proteomics, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Fission Yeast MAP Kinase Sty1 Is Recruited to Stress-induced Genes

doi: 10.1074/jbc.m710428200

Figure Lengend Snippet: FIGURE 2. Sty1 is recruited to the gpd1 promoter upon osmotic stress in a manner coincident with its own activation. A, ChIP assays showing recruit- ment of Sty1–6His2HA to the gpd1 promoter upon osmotic stress. Samples were prepared from sty1–6His2HA cells treated with 1 M sorbitol for the time points indicated (in minutes). The DNA recovered from the IP was assayed by PCR using primers specific to the gpd1, hmg1, and cdc2 promoters, the latter two of which are not induced upon stress. The control lanes show DNA ampli- fied from two different amounts of whole cell extracts (WCE) prior to perform- ing the IP. B, the kinetics of Sty1 activation were assayed by Western blotting of protein extracts prepared from the cells exposed to osmotic stress in A. The blots were probed with antibodies against the activation site of the p38 MAP kinase (anti-phospho-p38), which recognize the activation site of Sty1 when dually phosphorylated upon Thr171 and Tyr173. Total levels of Sty1 were assessed by re-probing the blot with anti-HA antiserum.

Article Snippet: Activated Sty1 was detected using the anti-phospho-p38 antiserum (Genway).

Techniques: Activation Assay, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Fission Yeast MAP Kinase Sty1 Is Recruited to Stress-induced Genes

doi: 10.1074/jbc.m710428200

Figure Lengend Snippet: FIGURE 5. Sty1 kinase activity is required for its recruitment to stress- responsive promoters. A, ChIP assays to assess the recruitment of Sty1 and Sty1 kinase-dead to stress-induced promoters. Assays were carried out as described in the legend to Fig. 3A using samples prepared from sty1-Pk and sty1kd-Pk cells that had been exposed to 1 M sorbitol or 2 mM H2O2 for 15 min. Cells grown without stress were used as a control (indicated by ). B, Western blot to examine the activation of Sty1 in the sty1 kinase-dead (sty1kd-Pk) strain compared with the sty1-Pk strain. Whole cell extracts were prepared before or after 1 M sorbitol stress. Sty1 activation was assessed using anti- phospho-p38 antiserum. The loading was assessed using anti-Pk antiserum.

Article Snippet: Activated Sty1 was detected using the anti-phospho-p38 antiserum (Genway).

Techniques: Activity Assay, Control, Western Blot, Activation Assay