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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 2. Effect of Rac1 siRNA and the Rac1 inhibitor NSC23766 on NOD2- and TLR2-mediated IL-8 secretion. Primary human monocytes were transfected with control nonsilencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed (A). Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B, Primary human monocytes were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP or Malp2 for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. THP-1 cells were untreated (none) or preincubated overnight with the Rac1 inhibitor NSC23766 (200 M). Subsequently, the cells were either stimulated with MDP (C) or Malp2 (D) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 3. Influence of Rac1 on NOD2- or TLR2-mediated IL-8 and NF-B activation. A, HEK293 cells were left untreated, or were trans- fected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed in duplicates. Membranes were simul- taneously probed with anti-ERK2 Abs to confirm equal protein loading. B–D, HEK293 cells were left untreated (ctrl), or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si- Rac1). After 48 h, the cells were additionally cotransfected with NOD2 (B and C) or TLR2 (D) expression plasmids, together with an IL-8- reporter (B) or NF-B reporter construct (C and D) and a -galactosi- dase reporter plasmid. Cells were either left untreated () or stimulated with MDP (B and C) or Malp2 (D) and relative luciferase activities were obtained. E and F, HEK293 cells seeded in 24-well plates were tran- siently transfected with a control vector (ctrl) or NOD2 expression plas- mid along with an IL-8 luciferase reporter plasmid (E) or a NF-B- driven luciferase reporter (F), respectively, and a -galactosidase reporter plasmid. Additionally, the cells were cotransfected with wild- type Rac1 (Rac1wt), dominant negative Rac1N17 or constitutively ac- tive Rac1L61. Cells were either stimulated with 10 g/ml MDP (MDP) or left untreated (), and relative luciferase activities were obtained the next day. G, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl), or a RIP2 expression plasmid along with a NF-B-driven luciferase reporter and a -galactosidase plasmid. The influence of Rac1 was tested by additionally introducing wild-type Rac1 (Rac1wt), dominant negative Rac1 (Rac1N17), or con- stitutive active Rac1 (Rac1L61) mean SD; , p 0.01.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Activation Assay, Control, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Luciferase, Dominant Negative Mutation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 7. Rac1 and -PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or -PIX siRNA, as indicated, and were stimulated with 10 g/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent im- munoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 6. Involvement of -PIX in NOD2-medi- ated signaling. MDP-stimulated primary monocytes or THP-1 cells (A and B) were lysed at different time points, immunoprecipitations of endogenous Rac1 (A) or endogenous NOD2 (B) with the respective Abs were performed, and immune complexes were probed for the presence of -Pix. Equal protein amounts in the lysates were confirmed by blotting total cell lysates with an ERK2 Ab (Input). All experiments were repeated three times. C, THP-1 cells were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting -Pix (si--Pix_S1 (sequence 1), si-Pix_S2 (sequence 2)). After 72 h, cells were lysed and Western blots using anti--Pix Abs were performed. Western blots were si- multaneously probed with anti-ERK2 Abs to confirm equal protein loading. D, THP-1 cells were transfected as indicated, incubated for 72 h, and stimulated with MDP (10 g/ml) for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti- NOD2 Abs. E and F, THP-1 cells were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP (E) or Malp2 (F) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data pre- sented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Control, Sequencing, Western Blot, Incubation, Membrane, Enzyme-linked Immunosorbent Assay

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

Journal: Naunyn-Schmiedeberg's Archives of Pharmacology

Article Title: The effects of melatonin on oxidative stress, inflammation, apoptosis and Nrf2/HO-1 in acrylamide-induced lung injury in rats

doi: 10.1007/s00210-025-04292-8

Figure Lengend Snippet: Lung TNF-α, IL-1β, IL-6, NF-κB, iNOS, COX-2, MPO, p38 MAPK, and IL-10 activities in experimental groups ( n = 10). Results are expressed as mean ± SEM

Article Snippet: Catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH), heme oxygenase 1 (HO-1), interleukin-1 beta (IL-1β), interleukin-10 (IL-10), malondialdehyde (MDA), nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear factor kappa (NF-κB), superoxide dismutase (SOD), tumor necrosis factor alpha (TNF-α), caspase 3 (CASP3), inducible nitric oxide synthase (iNOS; NOS2), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), p38 mitogen-activated protein kinases (p38-MAPK), and myeloperoxidase (MPO) enzyme-linked immunosorbent assay (ELISA) kits were purchased from BT-LAB.

Techniques: