Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: List of siRNA target sequences.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Sequencing

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: Antibodies used for the western blot.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Western Blot, Immunofluorescence, Immunoprecipitation

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: Production and function of TGF-beta in AECs. (a) ELISA for TGF-beta conducted on AEC supernatant that had been stimulated with CSE for 48 h. AECs showed a significantly increased production of TGF-beta when stimulated with CSE (P < .05) and NAC can inhibit this production (P < .05). A one-way ANOVA with the Tukey Multiple Comparisons Test was used to test statistical significance. (b) AECs treated with 2 ng/mL of TGF-beta for up to 1 h caused activation of p38MAPK (two-fold) compared to control treated AECs. (c) AECs treated with CSE for 1 h significantly induced p38MAPK activation (P < 0.0001) while TGF receptor antagonist prevented p38MAPK activation (P = 0.03). (d) ELISA for TGF-beta conducted on amniotic fluid of TNIL or TL deliveries showed significant expression of TGF-beta at term (P < .05). A one-tailed t-test was used to test statistical significance. (e) Immunofluorescence colocalized TAK (red) and TAB1 (green) inside control treated AECs. White lines represent regions of interest to look for TAK1-TAB1 colocalization. Both cells showed overlapping line graphs of TAK1-TAB1 documenting colocalization within the cytoplasm. Scale bar is set to 30 μM. (f) Western blot analysis confirmed that TAB1 can be significantly (P = .047) activated by CSE and antioxidant NAC can significantly prevent this activation (P < = .057). (g) Quantitative densitometry of P-TAB1 over actin for panel f showing statistical significance. A one-way ANOVA with the Tukey Multiple Comparisons Test was used to test statistical significance.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Expressing, One-tailed Test, Immunofluorescence, Western Blot

Journal: Biology of Reproduction

Article Title: Oxidative stress-induced TGF-beta/TAB1-mediated p38MAPK activation in human amnion epithelial cells †

doi: 10.1093/biolre/ioy135

Figure Lengend Snippet: Inhibition of TAK1 does not inhibit p38MAPK activation. (a) mRNA levels of TAK1 showed siRNA to TAK1 decrease its expression by 91% (P = .003) but NT siRNA does not decrease TAK1 gene expression 6% (P = .58). A two-tailed t-test was used to test statistical significance. (b) AECs treated with CSE and siRNA to TAK1 did not significantly reduce p38MAPK phosphorylation. (c) Quantitative densitometry (b) of P-p38MAPK over total p38MAPK showed significant increase of p38MAPK phosphorylation in AECs when treated with CSE (P = .001) and NT siRNA (P = .001); however, there was no significant difference of activated p38MAPK phosphorylation when treated with siRNA to TAK1 + CSE (P = 1.00). Surprisingly, NT siRNA + CSE also significantly upregulated p38MAPK activation compared to CSE alone (P = .006). A two-tailed t-test was used to test statistical significance.

Article Snippet: The following antihuman antibodies were used for western blot: ASK1 (1:750, Cell Signaling, Danvers, MA), TRX (1:1000, Abcam, Cambridge, United Kingdom), TAB1 (1:1000, R&D, Minneapolis, MN), P-TAB1 (1:800, Milipore, Thr431, Burlington, MA), P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling) (Table ). table ft1 table-wrap mode="anchored" t5 Table 3. caption a7 Protein Name Catalog Number Company Dilution Method ASK1 D11C9 Cell Signaling 1:750 Western Blot TRX Ab16965 Abcam 1:1000 Western Blot TAB1 AF3578 R&D 1:1000 Western Blot P-TAB1(Thr431) 06–1334 Milipore 1:800 Western Blot P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Western Blot p38 MAPK 9212S Cell Signaling 1:1000 Western Blot Β-Actin A5441 Sigma-Aldrich 1:20,000 Western Blot Cytokeratin-18 Ab668 Abcam 1:300 Immunofluorescence ASK1 Ab45178 Abcam 1:250 Immunofluorescence TRX Ab16965 Abcam 1:500 Immunofluorescence P-p38 MAPK (T180/y182) 9211L Cell Signaling 1:300 Immunofluorescence TAK1 Sc-7967 Santa Cruz 1:300 Immunofluorescence TAB1 AF3578 R&D 1:300 Immunofluorescence ASK1 D11C9 Cell Signaling 1:100 Immunoprecipitation TRX Ab16965 Abcam 1:100 Immunoprecipitation GSK3B mAb#9832 Cell Signaling 1:100 Immunoprecipitation Open in a separate window Antibodies used for the western blot.

Techniques: Inhibition, Activation Assay, Expressing, Two Tailed Test

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, 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, Activation Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Cytokine-Induced Muscle Atrophy and Weakness Can Be Ameliorated by an Inhibition of TGF-β-Activated Kinase-1

doi: 10.3390/ijms25115715

Figure Lengend Snippet: TNF-α and IL-1β enhanced TAK1 signaling, and LLZ abrogated TAK1 signaling in C2C12 cells. Western blot analysis of intracellular IκBα, phosphorylated TAK1, p38, and ERK in C2C12 cells treated with ( A ) TNF-α or ( B ) IL-1β in the presence or absence of LLZ.

Article Snippet: The following reagents were purchased from the indicated manufactures: mouse monoclonal antibodies against TNF-α and IL-1β, rabbit polyclonal antibodies against TAK1, IκBα, rabbit monoclonal antibodies against phosphorylated p38 MAPK, p38 MAPK, phosphorylate ERK, ERK, β-actin, horseradish peroxidase (HRP) anti-rabbit IgG, and anti-mouse IgG from Cell Signaling Technology (Beverly, MA, USA); antibodies against myosin heavy chain and goat IgG-FITC from Santa Cruz (Dallas, TX, USA); rabbit polyclonal anti-phosphorylated TAK1 from Cusabio (Houston, TX, USA); rabbit polyclonal antibodies against albumin from Invitrogen (Waltham, MA, USA); TAK1 inhibitor, LL-Z1640-2 (LLZ), from BioAustralis (Smithfield, NSW, Australia); recombinant human IL-1β from Wako (Osaka, Japan); recombinant human TNF-α from R&D Systems (Minneapolis, MN, USA); mannan from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Western Blot