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: 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: Cancer discovery

Article Title: p38MAPK plays a crucial role in stromal mediated tumorigenesis

doi: 10.1158/2159-8290.CD-13-0743

Figure Lengend Snippet: (A) Western blot analysis demonstrating that SB203580 treatment inhibits p38MAPK activity. As expected, SB203580 treatment significantly impacts phosphorylation of p38MAPK’s direct downstream target, Hsp27. Total Hsp27 was used as loading control.

Article Snippet: The primary antibodies used were: polyclonal AUF1 (Millipore, Billerica, MA, catalog number 07260MI) at 1:3000, polyclonal p-p38 (PhosphoSolutions, Aurora, CO, catalog number p190-1802) at 1:1000, polyclonal p38 (Cell Signaling, Boston, MA, catalog number 9218) at 1:1000, monoclonal β-catenin (BD Biosciences, San Jose, CA, catalog number 610153) at 1:5000, and monoclonal α-tubulin (Abcam, Cambridge, MA, product number ab6160) at 1:1000.

Techniques: Western Blot, Activity Assay, Phospho-proteomics, Control

Journal: Cancer discovery

Article Title: p38MAPK plays a crucial role in stromal mediated tumorigenesis

doi: 10.1158/2159-8290.CD-13-0743

Figure Lengend Snippet: (A) RNA-seq analysis was performed on young fibroblasts, senescent fibroblasts, and senescent fibroblasts treated with SB203580. RNA-seq results were analyzed to determine the number of factors upregulated in response to senescence (SASP factors) and the number of p38MAPK-dependent factors. These results were also analyzed for overlap with the expression profiles of breast cancer (BC)-associated stroma.

Article Snippet: The primary antibodies used were: polyclonal AUF1 (Millipore, Billerica, MA, catalog number 07260MI) at 1:3000, polyclonal p-p38 (PhosphoSolutions, Aurora, CO, catalog number p190-1802) at 1:1000, polyclonal p38 (Cell Signaling, Boston, MA, catalog number 9218) at 1:1000, monoclonal β-catenin (BD Biosciences, San Jose, CA, catalog number 610153) at 1:5000, and monoclonal α-tubulin (Abcam, Cambridge, MA, product number ab6160) at 1:1000.

Techniques: RNA Sequencing, Expressing

Journal: Translational Neuroscience

Article Title: Euxanthone inhibits traumatic spinal cord injury via anti-oxidative stress and suppression of p38 and PI3K/Akt signaling pathway in a rat model

doi: 10.1515/tnsci-2021-0012

Figure Lengend Snippet: Effect of Euxanthone on p-p38 protein expression in t-SCI-induced rats. (a) Quantification and (b) representative Western blot images of the effect of Euxanthone on p-p38 protein expression. GAPDH served as an internal control. The data are represented as the mean value ± standard error of mean. # p < 0.01 relative to sham group, * p < 0.05 relative to t-SCI model group, and $ p < 0.05 relative to t-SCI model group and Eux30 study group. t-SCI, traumatic spinal cord injury; Eux30 and Eux60, Euxanthone 30 and 60 mg/kg, respectively, in t-SCI-induced rats; p, phosphorylated.

Article Snippet: The membranes were then incubated for 12 h at 4°C with primary antibodies purchased from Bio-rad Laboratories, Shanghai, China, namely – Receptor activator of nuclear factor-κB ligand (RANKL) (MCA5971), toll-like receptor 4(TLR4) (AHP1822), Nuclear factor (NF)-κB (VPA00015), phosphorylated (p)-NF-κB (AHP1342), PI3K (AHP2224), Akt (VMA00253) and GAPDH (HCA272), p38 (AHP2410), and p-p38 (#9211, Cell Signaling Technology, Shanghai, China).

Techniques: Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Computational and In Vitro Analysis of Plumbagin’s Molecular Mechanism for the Treatment of Hepatocellular Carcinoma

doi: 10.3389/fphar.2021.594833

Figure Lengend Snippet: (A) Protein expression levels of LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK in SMMC-7721 cells measured by Western blot after treatment with 5 μM PL, 10 μM 3-MA autophagy inhibitor, 10 μM rapamycin autophagy agonist, 10 μM SB202190, and 10 μM SB203580 p38 MAPK inhibitor. (B) LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK were used as internal reference total proteins for gray value analysis. * p < 0.05, ** p < 0.01 *** p < 0.001, compared with the PL group. # p < 0.05, ## p < 0.01 ### p < 0.001 vs. the control group. (C) Protein expression levels of LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK in BEL-7404 cells measured by Western blot after treatment with 5 μM PL, 10 μM 3-MA autophagy inhibitor, 10 μM rapamycin autophagy agonist, 10 μM SB202190, and 10 μM SB203580 p38 MAPK inhibitor. (D) LC3-I, LC3-II, p38 MAPK, and p-p38 MAPK were used as internal reference total proteins for gray value analysis. * p < 0.05, ** p < 0.01 *** p < 0.001, compared with the PL group. # p < 0.05, ## p < 0.01 ### p < 0.001 vs. the control group.

Article Snippet: Plumbagin (PL) was purchased from Sigma-Aldrich (St. Louis, MO, United States) with purity ≥98%; the Acridine Orange (AO)/Ethidium bromide (EB) Double Stain Kit was from Solable Technology (Beijing, China); N-acetyl-l-cysteine, SB203580, and SB202190 were from Sigma-Aldrich (St. Louis, MO, United States); SC-79, MEK2206, 3-MA, and Z-VAD-FMK were from Selleck (Texas, United States); the BCA Protein Assay Kit, ROS Assay Kit, Annexin V-FITC Apoptosis Detection Kit and Cell lysis buffer for Western were all obtained from Beyotime Biotechnology (Shanghai, China); antibodies against Akt, phospho-Akt, mTOR, phospho-mTOR, p38 MAPK, phospho-p38 MAPK, PI3K, phospho-PI3K, LC3B, cleave-RP, and cleave-caspase 3 were from Cell Signaling Technology, Inc. (Boston, MA, United States); and β-p38 MAPK was purchased from Boster Technology (Wuhan, China).

Techniques: Expressing, Western Blot, Control