rabbit anti human phospho tak1 thr184 187  (Cell Signaling Technology Inc)

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

doi: 10.1152/ajpgi.00400.2014

Figure Lengend Snippet: Fig. 1. Transforming growth factor (TGF)--activated kinase 1 (TAK1) is overexpressed in fibrotic tissue of Crohn’s disease (CD) patients. A: Western blot analysis of TAK1 expression in ileal tissues obtained from normal ileum of patients (n 9) with right colon carcinoma or stenotic segments of CD patients (n 15) and relative densitometric analysis. B: Sirius Red staining of ileal sections obtained from normal ileum or stenotic segments of CD patients (scale bars, 75 m) and relative Sirius Red staining quantification by ImageJ. Data are reported as means SE. **P 0.01 and *P 0.05 vs. control (CTRL). CXCL8 (C) and TNF (D) levels in ileal tissues measured by ELISA. Data are reported as box and whiskers of 2.5 and 97.5 percentiles of 8 determinations for control and 11 for CD. ***P 0.001 vs. control. E: Pearson correlation analysis of TAK1 expression with ileal fibrotic areas, CXCL8, and TNF. Dashed lines show the 95% confidence band of the best fit line.

Article Snippet: Following 1 h incubation with PBS, containing 5% BSA (Sigma), membranes were incubated at 4°C for 16 h with rabbit anti-human TAK1, rabbit anti-human TAK1 phosphorylated form (pTAK1; Cell Signaling Technology, Danvers, MA), mouse anti-human -SMA, mouse anti-human -actin, mouse anti-human -tubulin (Sigma), or goat anti-lamin A/C (a kind gift of Dr. G. Alvisi, Department of Ta, annealing temperature; -sma, -smooth muscle actin; FW, forward; RV, reverse; Fam, 6-carboxyfluorescein; Tamra, tetramethylrhodamine; Col1 1, collagen1( )1; Tak1, transforming growth factor- -activated kinase 1; Timp1, tissue inhibitor of metalloproteinase-1.

Techniques: Western Blot, Expressing, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

doi: 10.1152/ajpgi.00400.2014

Figure Lengend Snippet: Fig. 2. TAK1 mRNA level correlates with fibrotic markers in the ileal mucosa of CD patients. A–D: collagen1()1 (COL1A1), tissue inhibitor of metalloproteinase-1 (TIMP1), TNF, and TAK1 mRNA levels, respectively, in biopsies of ileal mucosa of CD patients (n 17), healthy control subjects (n 14), and ulcerative colitis (UC) patients (n 9). Data are reported as box and whiskers of 2.5 and 97.5 percentiles. *P 0.05 and **P 0.01 vs. CD. E: Pearson correlation analysis of TAK1 mRNA with COL1A1, TIMP1, and TNF mRNA of CD patients (n 17). Dashed lines show the 95% confidence band of the best fit line.

Article Snippet: Following 1 h incubation with PBS, containing 5% BSA (Sigma), membranes were incubated at 4°C for 16 h with rabbit anti-human TAK1, rabbit anti-human TAK1 phosphorylated form (pTAK1; Cell Signaling Technology, Danvers, MA), mouse anti-human -SMA, mouse anti-human -actin, mouse anti-human -tubulin (Sigma), or goat anti-lamin A/C (a kind gift of Dr. G. Alvisi, Department of Ta, annealing temperature; -sma, -smooth muscle actin; FW, forward; RV, reverse; Fam, 6-carboxyfluorescein; Tamra, tetramethylrhodamine; Col1 1, collagen1( )1; Tak1, transforming growth factor- -activated kinase 1; Timp1, tissue inhibitor of metalloproteinase-1.

Techniques: Control

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

doi: 10.1152/ajpgi.00400.2014

Figure Lengend Snippet: Fig. 3. TAK1 is overexpressed in CD-derived intestinal mucosal myofibroblast (IMF). A: tissue sections of ileum obtained from control patients with right colon carcinoma (n 9) or stenotic segments of CD patients (n 15) were stained for TAK1. -Smooth muscle actin (-SMA) was used as a myofibroblast marker (scale bars, 23.8 m). B: Western blotting analysis of -SMA on ileal tissues obtained from normal ileum (n 9) or stenotic segments (n 15) of CD patients and relative densitometric analysis. C: Pearson correlation analysis of Western blot data of -SMA with TAK1 expression in stenotic segments. Dashed lines show the 95% confidence band of the best fit line. D: immunofluorescence analysis of TAK1 in IMF obtained from normal ileum controls (n 3) and CD (n 3; scale bars, 37.5 m). E: Western blotting analysis of TAK1 on IMF obtained from normal ileum or stenotic segments of CD patients and densitometric analysis. F: real-time quantitative PCR (qPCR) for TAK1 in IMF obtained from normal ileum (n 4) or stenotic segment of CD (n 4) patients. Data are reported as means SE. **P 0.05 and *P 0.01 vs. control.

Article Snippet: Following 1 h incubation with PBS, containing 5% BSA (Sigma), membranes were incubated at 4°C for 16 h with rabbit anti-human TAK1, rabbit anti-human TAK1 phosphorylated form (pTAK1; Cell Signaling Technology, Danvers, MA), mouse anti-human -SMA, mouse anti-human -actin, mouse anti-human -tubulin (Sigma), or goat anti-lamin A/C (a kind gift of Dr. G. Alvisi, Department of Ta, annealing temperature; -sma, -smooth muscle actin; FW, forward; RV, reverse; Fam, 6-carboxyfluorescein; Tamra, tetramethylrhodamine; Col1 1, collagen1( )1; Tak1, transforming growth factor- -activated kinase 1; Timp1, tissue inhibitor of metalloproteinase-1.

Techniques: Derivative Assay, Control, Staining, Marker, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

doi: 10.1152/ajpgi.00400.2014

Figure Lengend Snippet: Fig. 4. TAK1 activation in IMF of CD patients. A: Western blotting analysis of phosphorylated (p)TAK1 in specimens obtained from normal ileum (n 9) or stenotic segments of CD patients (n 15) and relative densitometric analysis. B: tissue sections of ileum obtained from control patients with right colon carcinoma (n 9) or stenotic segments of CD patients (n 15) were stained for pTAK1. -SMA was used as a myofibroblast marker (scale bars, 23.8 m). Arrows, pTAK1 -SMA myofibroblasts. C: Western blotting of pTAK1 in IMF derived from control subjects and CD patients (n 4–5 cell lines for each group) and relative densitometric analysis. D: immunocytochemical analysis of pTAK1 in control and CD-derived IMF (scale bars, 37.5 m; n 3 cell lines for each group). E: Western blotting analysis of pTAK1 on CD-derived IMF treated with 10 g/ml anti-TGF- neutralizing antibody (ab) for 3.5 h and relative densitometric analysis (n 4). Data are reported as means SE. ***P 0.001, **P 0.05, and *P 0.01 vs. control.

Article Snippet: Following 1 h incubation with PBS, containing 5% BSA (Sigma), membranes were incubated at 4°C for 16 h with rabbit anti-human TAK1, rabbit anti-human TAK1 phosphorylated form (pTAK1; Cell Signaling Technology, Danvers, MA), mouse anti-human -SMA, mouse anti-human -actin, mouse anti-human -tubulin (Sigma), or goat anti-lamin A/C (a kind gift of Dr. G. Alvisi, Department of Ta, annealing temperature; -sma, -smooth muscle actin; FW, forward; RV, reverse; Fam, 6-carboxyfluorescein; Tamra, tetramethylrhodamine; Col1 1, collagen1( )1; Tak1, transforming growth factor- -activated kinase 1; Timp1, tissue inhibitor of metalloproteinase-1.

Techniques: Activation Assay, Western Blot, Control, Staining, Marker, Derivative Assay

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

doi: 10.1152/ajpgi.00400.2014

Figure Lengend Snippet: Fig. 6. TAK1 activation mediates the fibrogenic phenotype of IMF. IMFs, isolated from ileal tissues of control subjects (n 3) and CD patients (n 3), were treated with TGF-1, LPS, or MDP in the presence or absence of TAK1 inhibitor 5Z-7-oxozeaenol for 12 h in the case of mRNA analysis or 24 h for cytokine quantifications (n 3). IL-6 (A) and CXCL8 (B) levels were assessed by ELISA in culture supernatants. Data are reported as means SE. *P 0.01 vs. untreated. C: COL1A1 mRNA expression was measured by real-time qPCR. Data are reported as means SE. *P 0.01 vs. untreated control IMF.

Article Snippet: Following 1 h incubation with PBS, containing 5% BSA (Sigma), membranes were incubated at 4°C for 16 h with rabbit anti-human TAK1, rabbit anti-human TAK1 phosphorylated form (pTAK1; Cell Signaling Technology, Danvers, MA), mouse anti-human -SMA, mouse anti-human -actin, mouse anti-human -tubulin (Sigma), or goat anti-lamin A/C (a kind gift of Dr. G. Alvisi, Department of Ta, annealing temperature; -sma, -smooth muscle actin; FW, forward; RV, reverse; Fam, 6-carboxyfluorescein; Tamra, tetramethylrhodamine; Col1 1, collagen1( )1; Tak1, transforming growth factor- -activated kinase 1; Timp1, tissue inhibitor of metalloproteinase-1.

Techniques: Activation Assay, Isolation, Control, Enzyme-linked Immunosorbent Assay, Expressing

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

doi: 10.1152/ajpgi.00400.2014

Figure Lengend Snippet: Fig. 7. TAK1 is essential for the profibrogenic phenotype of IMF. A: percentage of TAK1 mRNA expression after 72 h transduction with an adenoviral vector expressing shRNA targeting TAK1 (pAdshTAK1) or the gene encoding -galactosidase (pAdshLacZ) as control (n 3). B: Western blotting of TAK1 after 72 h transduction with pAdshTAK1 or pAdshLacZ and relative densitometric analysis. Data are reported as means SE of triplicate determinations of experiments performed with IMF, derived from 3 different CD patients. *P 0.05 and **P 0.01 vs. pAdshTAK1. C: real-time qPCR for COL1A1 mRNA in control- and CD-derived IMF transduced with pAdshLacZ or pAdshTAK1 for 72 h and stimulated or not with TGF-1 for 12 h (n 3 for each group). Data are reported as means SE. *P 0.01 vs. untreated pAdshLacZ control IMF; §P 0.05 and §§P 0.01 vs. untreated pAdshLacZ CD IMF; °P 0.05 vs. TGF-1 pAdshLacZ control IMF. D: real-time qPCR for COL1A1 mRNA in control- and CD-derived IMF (n 3 for each group) after 36 h treatment with 5Z-7-oxozeaenol. E: [3H]proline incorporation in control- and CD-derived IMF (n 3 for each group) and culture medium after 36 h of treatment with 5Z-7-oxozeaenol. Data are reported as means SE. *P 0.05 and **P 0.01 vs. untreated ctrl IMF; §P 0.05 vs. untreated CD IMF; °P 0.05 vs. TGF-1 control IMF. F: Western blotting of -SMA expression in CD-derived IMF after 6 days of transduction with pAdshTAK1 or pAdshLacZ as control (n 3). G: representative images of immunofluorescence analysis of TAK1 and -SMA expression in CD IMF after 6 days transduction with pAdshTAK1 or pAdshLacZ as control (n 3; scale bars, 37.5 m).

Article Snippet: Following 1 h incubation with PBS, containing 5% BSA (Sigma), membranes were incubated at 4°C for 16 h with rabbit anti-human TAK1, rabbit anti-human TAK1 phosphorylated form (pTAK1; Cell Signaling Technology, Danvers, MA), mouse anti-human -SMA, mouse anti-human -actin, mouse anti-human -tubulin (Sigma), or goat anti-lamin A/C (a kind gift of Dr. G. Alvisi, Department of Ta, annealing temperature; -sma, -smooth muscle actin; FW, forward; RV, reverse; Fam, 6-carboxyfluorescein; Tamra, tetramethylrhodamine; Col1 1, collagen1( )1; Tak1, transforming growth factor- -activated kinase 1; Timp1, tissue inhibitor of metalloproteinase-1.

Techniques: Expressing, Transduction, Plasmid Preparation, shRNA, Control, Western Blot, Derivative Assay