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 medicine reports

Article Title: Fluoxetine protects against methamphetamine‑induced lung inflammation by suppressing oxidative stress through the SERT/p38 MAPK/Nrf2 pathway in rats.

doi: 10.3892/mmr.2016.6072

Figure Lengend Snippet: Figure 4. SERT protein expression and P‑38 MAPK phosphorylation in rat lungs in the different groups. (A) SERT protein expression and (B) the rela tive p‑p38 MAPK expression in lungs were determined and quantified. The data are expressed as the mean ± standard deviation (n=6; *P<0.05, **P<0.01 vs. the control group; +P<0.05 vs. the MA group). SERT, serotonin trans porter; MAPK, mitogen activated protein kinases; MA, methamphetamine; F2, fluoxetine at 2 mg/kg; F10, fluoxetine at 10 mg/kg.

Article Snippet: The membranes were probed with primary mouse monoclonal anti-β‐actin (1:2,000; cat. no. sc‐130300; Santa Cruz Biotechnology, Inc., Dallas, USA) and α‐tubulin 1:2,000; cat. no. 66031; ProteinTech Group, Inc., Chicago, IL, USA) and rabbit polyclonal anti‐SERT (1:200; cat. no. bs 1893R), IL‐6 (1:200; cat. no. bs 0379R), tumor necrosis factor‐α (TNF-α; 1:200; cat. no. bs 0078R), human heme oxygenase‐1 (HO‐1; 1:400; cat. no. bs 2075R) (Biosynthesis Biotechnology Co. Ltd.), anti‐Nrf2 (1:600; cat. no. 16396‐1‐AP; ProteinTech Group, Inc.), p38 (1:600; cat. no. ZS‐7149), and phosphorylated (p)‐p38 (1:600; cat. no. ZS‐101759; Zhongshan Golden Bridge Biotechnology Co., Ltd.) overnight at 4 ̊C.

Techniques: Expressing, Phospho-proteomics, Standard Deviation, Control

Journal: Cancer Research

Article Title: Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

doi: 10.1158/0008-5472.can-08-3797

Figure Lengend Snippet: Figure 5. Cytoskeleton and VEGF-dependent signaling in cultured endothelial cells isolated from Shb knockout or wild-type liver. A, the cytoskeleton of isolated endothelial cells maintained in tissue culture for 5 to 7 d was visualized by staining with rhodamine-phalloidin, showing a less regular shape with numerous extensions in Shb null cells. B, corresponding wild-type control. No clear cytoskeletal difference between wild-type or knockout cells was noted after stimulation with VEGF-A (C, Shb knockout; D, Shb wild type). Addition of VEGF to the control cells (D) produced changes that made the cells resemble the Shb null cells in the absence of VEGF-A. Horizontal scale bar is shown. Equal amounts of protein from the experimental groups (+/, 20 ng/mL VEGF-A for 2 min) were subjected to Western blot analysis for the phosphorylated proteins indicated. Shb blot shows unstimulated cells only. The blots were subjected to densitometric analysis for phosphorylated FAK, total FAK, phosphorylated p38, total p38, pMLC, phosphorylated ERK, and total ERK in three separate experiments. Quantitation of the relative phosphorylation of FAK, MLC, p38, and ERK is also given. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01 (paired Students’ t test).

Article Snippet: The samples were electrophoresed on SDS-polyacrylamide gels, protein was transferred on to Hybond-P filters (GE Healthcare), and these were then probed for pY-1175 VEGFR-2, total VEGFR-2, pY-397 FAK, total FAK, phosphorylated extracellular signal-regulated kinase (ERK), total ERK, and phosphorylated Akt, total Akt, phosphorylated myosin light chain (pMLC), pY-658 VE-cadherin, phosphorylated p38, and total p38 (all antibodies were from Cell Signaling, except for total VEGFR-2 from R&D Systems, pY-397 FAK from Biosource, pY-VE-cadherin from ProSci, and pMLC from Santa Cruz Biotechnology) before incubation with secondary antibodies and enhanced chemiluminescence.

Techniques: Cell Culture, Isolation, Knock-Out, Staining, Control, Produced, Western Blot, Quantitation Assay, Phospho-proteomics

Journal: Molecular medicine reports

Article Title: Helicobacter pylori infection enhances heparanase leading to cell proliferation via mitogen‑activated protein kinase signalling in human gastric cancer cells.

doi: 10.3892/mmr.2018.9558

Figure Lengend Snippet: Figure 2. H. pylori infection mediates the HPA protein expression increase in MKN‑45 cells via the MAPK signalling pathway. (A) Western blotting was used to detect the expression levels of p‑p38 MAPK, p38 MAPK, p‑p65 NF‑κB and p65 NF‑κB following different co‑culture durations. (B) p‑p38 MAPK protein expression. (C) p‑p65 NF‑κB protein expression levels were quantitatively analysed using densitometry. (D) Western blots and (E) quantitatively analysed expression of NF‑κB in MKN‑45 cells pre‑treated with SB203580 prior to co‑culture with H. pylori. *P<0.05 and **P<0.01, as indicated. MAPK, mitogen‑activated protein kinase; NF‑κB, nuclear factor‑κB; p‑, phosphorylated; H. pylori or Hp, Helicobacter pylori.

Article Snippet: Following the blocking step, the membranes were incubated with the following primary antibodies: anti-HPA1 (1:1,000; cat. no. ab128931), anti-phosphorylated (p)-p38 MAPK (1:1,000; cat. no. ab195049), anti-p38 MAPK (1:1,000; cat. no. ab170099), anti-p-p65 NF-κB (1:1,000; cat. no. ab76302), anti-p65 NF-κB (1:1,000; no. ab32536; all Abcam, Cambridge, UK) and anti-β-actin (1:2,000; cat. no. TA-09; OriGene Technologies, Inc., Beijing, China).

Techniques: Infection, Expressing, 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: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 8. Effects of GADD45a on the olaquindox-induced MAPKs pathways (A) HepG2 and HepG2-iGADD45a cells were exposed to 0, 200, 400 and 800 µg/mL olaquindox for 24 h. Expression of p-JNK/JNK, p-p38/p38, p-ERK/ERK was detected by western blotting. GAPDH was used for loading control. The densitometric analysis results of p-JNK/JNK, p-p38/p38 and p-ERK/ERK were shown on (B–D). All data and results were represented as means ± SD from three or more independent experiments. (* p < 0.05, ** p < 0.01, compared with the control; # p < 0.05, ## p < 0.01, compared to HepG2 groups).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Expressing, Western Blot, Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 9. Effects of JNK/p38 pathways on olaquindox-induced cell death. HepG2 cells were pretreated with SP600125 or SB203580 for 1 h, followed to replace with or without olaquindox at the final concentration of 400 µg/mL for additional 24 h. (A) Cell viability was measured by MTT method; (B) Morphologic observation (400×); (C) Cell apoptosis was measured by staining with Hoechst 33342 (400×); (D) Quantification of apoptosis in HepG2 cells. Bright blue cells were counted as apoptotic cells from five independent microscopic fields. Results were presented as mean ± SD, from three independent experiments. (* p < 0.05, compared to the olaquindox alone group).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Concentration Assay, Staining

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 10. JNK/p38 pathways played protect role in olaquindox-induced DNA damage. (A) HepG2 cells were treated with olaquindox (400 µg/mL) for 4 h after preincubation with SP600125 or SB203580 for 1 h; (B) % tail DNA; (C) tail length; (D) tail moment; (E) HepG2 cells were treated with olaquindox (400 µg/mL) for 24 h after preincubation with SP600125 or SB203580 for 1 h. Cells were observed under a Leica inverted fluorescence microscope (400×). 1000 binucleated cells were recorded from each experiment and three independent experiments were carried out. All results were presented as mean ± SD. (** p < 0.01, compared with the control group; # p < 0.05, compared to olaquindox group).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Microscopy, Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 11. JNK/p38 pathways inhibited olaquindox-induced S-phase arrest. (A) HepG2 cells were treated with 400 µg/mL of olaquindox for 24 h after preincubation with SP600125 or SB203580 for 1 h; (B) Effect of HepG2 cells after preincubation with SP600125 or SB203580 on cell cycle distribution; (C) Effect of HepG2 cells treated with 400 µg/mL of olaquindox for 24 h after preincubation with SP600125 or SB203580 for 1 h on cell cycle distribution. Samples were measured by a flow cytometer. A minimum of 20,000 cells were recorded from each experiment. All results were presented as mean ± SD and three independent experiments were carried out. (** p < 0.01, compared with the control; # p < 0.05, compared to HepG2 groups).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Cytometry, Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 12. The relationship between GADD45a and JNK/p38 pathways in olaquindox response in the HepG2 cells. (A,B) Effects of SP600125 and SB203580 on the protein level of GADD45a in HepG2 cells. Cells were pre-treated with these inhibitors for 1 h before olaquindox treatment; (C) Effects of SP600125 and SB203580 on the cell viability both in HepG2 and HepG2-iGADD45a cells. All data and results were represented as means ± SD from three or more independent experiments. (* p < 0.05, ** p < 0.01, compared with the control; # p < 0.05, compared to HepG2 groups).

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Control

Journal: Molecules

Article Title: GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

doi: 10.3390/molecules22010124

Figure Lengend Snippet: Figure 13. A schematic diagram of the protective effect of GADD45a on olaquindox induced DNA damage and cell cycle arrest in HepG2 cells. Olaquindox could induce the exceeding reactive oxygen species (ROS) in the metabolic process, subsequently induced the DNA damage and activated GADD45a and JNK/p38 pathways. The activation of JNK/p38 may play a protective role in olaquindox induced cell death, DNA damage and cell cycle arrest. GADD45a could effective inhibit olaquindox induced DNA damage and S-phase arrest in HepG2 cell and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest.

Article Snippet: Mouse monoclonal antibody against p38 (1:1000) and phospho-p38 (1:1000), rabbit monoclonal antibody against JNK (1:1000), phospho-JNK (1:1000), ERK (1:1000) and phospho-ERK (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). p21 (1:1000), p53 (1:1000), GAPDH (1:1000) and β-actin (1:1000) were obtained Molecules 2017, 22, 124 16 of 19 from Zhongshan Golden Bridge (Beijing, China).

Techniques: Activation Assay

Journal: Endocrinology

Article Title: Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6

doi: 10.1210/en.2013-1822

Figure Lengend Snippet: Activation of MAPK in endometrial cells treated with lipopeptide PAMPs. Endometrial epithelial cells (A) and stromal cells (B) were collected 0, 5, 10, 15, 20, or 25 minutes after treatment with 100 ng/mL PAM or 100 ng/mL FSL-1. The protein from the cells was analyzed by SDS-PAGE and immunoblotted with antibodies against total and phosphorylated forms of p38 (t-p38 and p-p38) and ERK1/2 (tERK1/2 and pERK1/2; □, tERK2; ■, pERK2), and α-tubulin as visual confirmation of the precision of protein loading and transfer. The image for each cell type is representative of 3 independent experiments for PAM (left panel) or FSL-1 (right panel), and the histograms represent the mean ± SEM of densitometric analysis of the ratio of phosphorylated p-p38 to t-p38, pERK1 to tERK1 or pERK2 to tERK2, expressed as fold activation compared with time 0. Values differ from time 0 when data were analyzed by ANOVA, using the Dunnett pairwise multiple comparison t test: *, P < .05.

Article Snippet: Membranes were probed with antibodies targeting total and phosphorylated forms of ERK1/2 (anti-ERK1/2 [AB17942; [Abcam] and anti-MAPK activated diphosphorylated ERK1/2 [M8159; Sigma-Aldrich]), p38 (MAPK p38/MAPK14 [AP03041SU-N; Acris antibodies, 2B Scientific] and MAPK p38/MAPK14pThr180/pTyr182 [AP05898PU-N, Acris antibodies, 2B Scientific]), and NFκB (NFκB p65 [4764S; New England Biolabs] and phospho NFκB p65(Ser536) [3033L; New England Biolabs]), with protein loading confirmed using antibodies against α-tubulin (bovine α-tubulin, A11126; Invitrogen) or β-actin (bovine anti-β actin, ab8226; Abcam); the antibodies are further detailed in Supplemental Table 1 and were selected on the basis of recognition of immunoreactive proteins of appropriate molecular weight and previous publications ( , , ).

Techniques: Activation Assay, SDS Page, Comparison

Journal: Endocrinology

Article Title: Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6

doi: 10.1210/en.2013-1822

Figure Lengend Snippet: Attenuation of endometrial cell responses to lipopeptides by inhibition of MAPK. Endometrial epithelial (A and B) or stromal cells (C and D) were treated for 30 minutes in medium containing vehicle (V), ERK1/2 inhibitor (ERKi) (ERK activation inhibitor peptide I, 10 μM) or p38 inhibitor (p38i) (InSolution SB 203580, 10 μM) and then were cultured in the same treatment for 6 hours in control medium or medium containing 100 ng/mL PAM (A and C) or 100 ng/mL FSL-1 (B and D). Supernatants were harvested to measure the accumulation of IL-6 by ELISA, and results are expressed as a percentage of treatment with PAM (A and C) or FSL-1 (B and D). Data are presented as mean + SEM percentages and represent 3 independent experiments. Values differ from those for PAMP when data were analyzed by ANOVA using the Dunnett pairwise multiple comparison t test: *, P < .05.

Article Snippet: Membranes were probed with antibodies targeting total and phosphorylated forms of ERK1/2 (anti-ERK1/2 [AB17942; [Abcam] and anti-MAPK activated diphosphorylated ERK1/2 [M8159; Sigma-Aldrich]), p38 (MAPK p38/MAPK14 [AP03041SU-N; Acris antibodies, 2B Scientific] and MAPK p38/MAPK14pThr180/pTyr182 [AP05898PU-N, Acris antibodies, 2B Scientific]), and NFκB (NFκB p65 [4764S; New England Biolabs] and phospho NFκB p65(Ser536) [3033L; New England Biolabs]), with protein loading confirmed using antibodies against α-tubulin (bovine α-tubulin, A11126; Invitrogen) or β-actin (bovine anti-β actin, ab8226; Abcam); the antibodies are further detailed in Supplemental Table 1 and were selected on the basis of recognition of immunoreactive proteins of appropriate molecular weight and previous publications ( , , ).

Techniques: Inhibition, Activation Assay, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Comparison