primary antibodies against phopho-smad2/3 (psmad2/3, 1:1000)  (Cell Signaling Technology Inc)

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Critical Role for Transglutaminase 2 in Scleroderma Skin Fibrosis and in the Development of Dermal Sclerosis in a Mouse Model of Scleroderma

doi: 10.1002/art.43104

Figure Lengend Snippet: Increased expression of TGM2 by SSc fibroblasts and inhibition of TGM2 attenuates the expression of fibrotic protein markers in SSc dermal fibroblasts in vitro. (A) Expression of TGM2 (IA12 antibody) in primary HC fibroblasts (n = 6) and scleroderma fibroblasts (n = 6) was analyzed by Western blotting and normalized to expression of GAPDH. (B) TGM2 expression in primary HC fibroblasts (n = 3) was determined following fibroblast treatment with TGF‐β1 (4 ng/mL) for 24 hours. (C) Dermal fibroblasts isolated from HC and patients with SSc were cultured with TGM2 neutralizing antibody BB7.BB over a dose response range 0–1250 nM. (D) Survey of three HC and scleroderma fibroblasts cell lines culture in the presence of 1000 nM of BB7.BB. Expression of Col‐1, αSMA, and CCN2 were analyzed by Western blotting and normalized to expression of GAPDH. Densitometry analysis of Western blots (D; right) is shown. Bars show mean ± standard error of the mean. Statistical significance was tested using the t ‐test, * P < 0.05. HC, healthy control; SEM, scanning electron microscopy; SSc, scleroderma; TGF, transforming growth factor; TGM2, transglutaminase 2.

Article Snippet: Protein bands were transferred onto nitrocellulose membranes (GE Healthcare), which were incubated in block buffer (5% nonfat dry milk, 0.1% Tween‐20 [Sigma] in phosphate‐buffered saline) for 1 hour, followed by incubation at 4°C overnight with primary antibodies against TGM2 (IA12, UCB Pharma), α‐SMA (71 ng/mL, Dako; Cat M0851/1A4), Col‐1 (0.4 μg/mL, Millipore; Cat AB78), CCN2 (0.1 μg/mL, Santa Cruz Biotechnology; Cat SC‐14939), TNC2 μg/mL (Proteintech; Cat 67710‐1), Shad 2 (Cell signaling; Cat 5678), pSmad2/3 (Cell signaling; Cat 8828), and GAPDH (0.2 μg/mL, Abcam; Cat Ab8245/6C5), diluted in block buffer.

Techniques: Expressing, Inhibition, In Vitro, Western Blot, Isolation, Cell Culture, Control, Electron Microscopy

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Critical Role for Transglutaminase 2 in Scleroderma Skin Fibrosis and in the Development of Dermal Sclerosis in a Mouse Model of Scleroderma

doi: 10.1002/art.43104

Figure Lengend Snippet: TG2 antibody attenuates extracellular matrix deposition of fibroblasts in vitro. In (A), the readout is mean total intensity for each fluorophore. Data for fibronectin (left), collagens I and III (middle), and collagen IV (right), compare seven cultures derived from HC and seven from patients with SSc. In (B), cells were culture as in (A), but in the presence of BB7, and matrix was stained with antibodies to fibronectin (green) and collagens I, III (purple), and IV (blue). The data show responses from four cultures derived from HC and four from patients with SSc run in 2 independent experiments in the presence of BB7 (10 nm or 1000 nM) relative to 1000nM IgG control and given as percentage reduction in the amount of deposited extracellular matrix. ³¹ All images shown are representative merged images. Statistical significance was determined by t ‐test * P = 0.05, *** P < 0.001. Scale bar = 100 nm. HC, healthy control; SSc, systemic sclerosis. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.43104/abstract .

Article Snippet: Protein bands were transferred onto nitrocellulose membranes (GE Healthcare), which were incubated in block buffer (5% nonfat dry milk, 0.1% Tween‐20 [Sigma] in phosphate‐buffered saline) for 1 hour, followed by incubation at 4°C overnight with primary antibodies against TGM2 (IA12, UCB Pharma), α‐SMA (71 ng/mL, Dako; Cat M0851/1A4), Col‐1 (0.4 μg/mL, Millipore; Cat AB78), CCN2 (0.1 μg/mL, Santa Cruz Biotechnology; Cat SC‐14939), TNC2 μg/mL (Proteintech; Cat 67710‐1), Shad 2 (Cell signaling; Cat 5678), pSmad2/3 (Cell signaling; Cat 8828), and GAPDH (0.2 μg/mL, Abcam; Cat Ab8245/6C5), diluted in block buffer.

Techniques: In Vitro, Derivative Assay, Staining, Control

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Critical Role for Transglutaminase 2 in Scleroderma Skin Fibrosis and in the Development of Dermal Sclerosis in a Mouse Model of Scleroderma

doi: 10.1002/art.43104

Figure Lengend Snippet: TGM2 inhibition attenuates TGF‐β‐induced expression of fibrotic protein markers and alters Smad 2/3 phosphorylation and TGF‐β levels in control and SSc dermal fibroblasts. (A) Dermal fibroblasts isolated from HC (n = 3) and SSc cell lines (n = 3) were cultured alone or with recombinant TGF‐β1 (4 ng/mL), and then treated with combinations of control IgG, antibody BB7 (anti‐TGM2; 1000 nM), a pan‐TGF‐β blocking antibody (10 ug/mL), or in the presence of a small‐molecule inhibitor of ALK5inh/TGF‐βRI (SB431542; 10uM) for a further 48 hours. Expression of Col‐1, TNC, and αSMA were analyzed by Western blotting; blots shown are representative of n = 3. (B) Levels of SMAD2/3, phospho‐SMAD2/3, collagen I, and αSMA were assessed in SSc cell lines cultured alone or treated with BB7, a pan‐TGF‐β blocking antibody or with the ALK5 inhibitor. (C) Primary SSc dermal fibroblasts were grown in culture and then treated for 48 hours with either of BB7 or control IgG (1000nM). The media was then removed and the level of active TGF‐β measured using the mink lung cell bioassay. Data represent mean ± standard error of the mean for luminescence. * P < 0.05. HC, healthy control; SSc, scleroderma; TGF, transforming growth factor; TGM2, transglutaminase 2. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.43104/abstract .

Article Snippet: Protein bands were transferred onto nitrocellulose membranes (GE Healthcare), which were incubated in block buffer (5% nonfat dry milk, 0.1% Tween‐20 [Sigma] in phosphate‐buffered saline) for 1 hour, followed by incubation at 4°C overnight with primary antibodies against TGM2 (IA12, UCB Pharma), α‐SMA (71 ng/mL, Dako; Cat M0851/1A4), Col‐1 (0.4 μg/mL, Millipore; Cat AB78), CCN2 (0.1 μg/mL, Santa Cruz Biotechnology; Cat SC‐14939), TNC2 μg/mL (Proteintech; Cat 67710‐1), Shad 2 (Cell signaling; Cat 5678), pSmad2/3 (Cell signaling; Cat 8828), and GAPDH (0.2 μg/mL, Abcam; Cat Ab8245/6C5), diluted in block buffer.

Techniques: Inhibition, Expressing, Phospho-proteomics, Control, Isolation, Cell Culture, Recombinant, Blocking Assay, Western Blot, Bioassay

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Critical Role for Transglutaminase 2 in Scleroderma Skin Fibrosis and in the Development of Dermal Sclerosis in a Mouse Model of Scleroderma

doi: 10.1002/art.43104

Figure Lengend Snippet: TGM2 gene deletion protects mice from bleomycin‐induced skin fibrosis and impacts upon primary dermal fibroblast functional activities. In (A), skin sections were stained with Masson's Trichrome and picrosirius red and dermal thickness measured using the NanoZoomer NDP software. Three measurements were taken across the skin sections and the averages skin thickness given as fold change relative to saline treated control mice. Stained sections were scanned using a NanoZoomer and viewed with the NDP viewer (×10 magnification). Data are presented as fold change in dermal thickness. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Bar = 200 microns. (B) Primary dermal fibroblasts were culture to confluence, and the monolayers scratched to induce a single injury in the monolayer. A zero time point following wounding (left), scratch repair after 48 hours for WT (middle), and TG2 null (right) fibroblast populations are shown. The upper panel shows migration of fibroblast after 48 hours in the absence and presence (lower panels) of TGF‐β. (C) Dermal biopsies (4 mm) were taken from 6 to 10 mice and snap frozen ready for use. Collagen content was measured using the Sircoll as per the manufacturer's instructions. The Sircoll assay is a colorimetric assay for tissue collagen against a curve of collagen reference standards. Measurements are presented as fold change relative to the saline treated controls **** P = 0.0001. Average collagen content per biopsy were WT/saline (n = 6/27.3 ug), WT/bleomycin (n = 8/41.9ug), TG2 null/saline (n = 10/40.8 ug), and TG2 null/bleomycin (n = 10/43.95 ug). Fibroblast populations derived from explant skin cultures, which were placed with 3D collagen gels (left panel), and their level of contraction was assessed at 48 hours (right panel). Contraction was determined by quantification of gel weight after contraction. Statistical significance was tested by one way ANOVA with Tukey's multiple comparison, * P < 0.05, *** P < 0.0001. Scale bar = 200 μm. ANOVA, analysis of variance; KO, knockout; TGM2, transglutaminase 2; TGF, transforming growth factor; WT, wild type. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.43104/abstract .

Article Snippet: Protein bands were transferred onto nitrocellulose membranes (GE Healthcare), which were incubated in block buffer (5% nonfat dry milk, 0.1% Tween‐20 [Sigma] in phosphate‐buffered saline) for 1 hour, followed by incubation at 4°C overnight with primary antibodies against TGM2 (IA12, UCB Pharma), α‐SMA (71 ng/mL, Dako; Cat M0851/1A4), Col‐1 (0.4 μg/mL, Millipore; Cat AB78), CCN2 (0.1 μg/mL, Santa Cruz Biotechnology; Cat SC‐14939), TNC2 μg/mL (Proteintech; Cat 67710‐1), Shad 2 (Cell signaling; Cat 5678), pSmad2/3 (Cell signaling; Cat 8828), and GAPDH (0.2 μg/mL, Abcam; Cat Ab8245/6C5), diluted in block buffer.

Techniques: Functional Assay, Staining, Software, Saline, Control, Migration, Colorimetric Assay, Derivative Assay, Comparison, Knock-Out