primary antibodies against col-1  (Millipore)

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

Journal: Scientific Reports

Article Title: Downregulation of enhancer RNA AC003092.1 is associated with poor prognosis in kidney renal clear cell carcinoma

doi: 10.1038/s41598-024-64431-8

Figure Lengend Snippet: Slowing down of ECM process after knockdown of AC003092.1. ( A–C ) Immunofluorescence shows that FN and Col-1 expression is reduced after knockdown of AC003092.1. ( D,E ) Western blotting experiments showed that FN and Col-1 protein expression levels were reduced after knockdown of AC003092.1.

Article Snippet: The samples were subsequently incubated with primary antibodies against Col-1 (GB114197-100, Servicebio, China), FN (GB114491-100, Servicebio, China), and P53 (GB15627-100, Servicebio, China), followed by a goat anti-rabbit conjugate (GB25303, Servicebio, China) as the secondary antibody.

Techniques: Immunofluorescence, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Rutaecarpine ameliorates osteoarthritis by inhibiting PI3K/AKT/NF-κB and MAPK signalling transduction through integrin αVβ3

doi: 10.3892/ijmm.2023.5300

Figure Lengend Snippet: Protective effects of RUT against IL-1β-induced inflammation and extracellular matrix degradation in mouse chondrocytes. The cells were exposed to IL-1β (5 ng/ml) with/without RUT (1, 2.5, 5 and 10 μ M) for 24 h. (A and B) The protein expression levels of MMP3, MMP13, IL-1β, COX2, IL-6, TNF-α, SOX9, COL II and aggrecan were determined using western blot analysis. (C-G) Quantification analysis of the results of western blotting. (D and E) Relative mRNA expression levels of IL-6 and TNF-α in chondrocytes stimulated with IL-1β (5 ng/ml) and treated with or without RUT (1, 2.5, 5 and 10 μ M) for 24 h. The values are presented as the mean ± SD of three independent experiments. # P<0.05 vs. control group; ** P<0.01, *** P<0.001 and **** P<0.0001 vs. IL-1β group. (H) The protein expression of COL II was detected using immunofluorescence following treatment of the cells with IL-1β (5 ng/ml) with/without RUT (10 μ M) for 24 h. IL-1β, interleukin 1 β; RUT, rutaecarpine; MMP, matrix metalloproteinase; COX2, cyclooxygenase 2; SOX9, SRY-box transcription factor 9; COL II, collagen type II.

Article Snippet: Subsequently, primary antibodies against COL II (1:200; cat. no. 28459-1-AP, Proteintech Group, Inc.) and p65 (1:400; cat. no. 8242, Cell Signaling Technology, Inc.) were used to incubate the cells at 4°C overnight.

Techniques: Expressing, Western Blot, Control, Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: Rutaecarpine ameliorates osteoarthritis by inhibiting PI3K/AKT/NF-κB and MAPK signalling transduction through integrin αVβ3

doi: 10.3892/ijmm.2023.5300

Figure Lengend Snippet: The beneficial effects of RUT on IL-1β-stimulated chondrocytes were attenuated following integrin αVβ3 knockdown. (A and B) Relative mRNA levels of ItgαV and Itgβ3 in chondrocytes exposed to 5 ng/ml IL-1β with/without RUT (1, 2.5, 5 and 10 μ M) for 24 h. The knockdown efficiency of ItgαV and Itgβ3 siRNA transfection was verified using RT-qPCR. (C-E and H-M) The expression levels of inflammation and extracellular matrix degradation-related proteins were determined using western blot analysis; the outcomes of the chondrocytes were quantified in the presence/absence of 5 ng/ml IL-1β, RUT (10 μ M) and ItgαVβ3 for 24 h. (F and G, and N-S) Western blot analysis and quantification analysis were used to measure the levels of PI3K/Akt/NF-κB and MAPK-related signalling proteins in IL-1β (5 ng/ml)-stimulated chondrocytes for 30 min following transfection with/without ItgαVβ3 siRNA and RUT (10 μ M) for 24 h. Data are presented as the mean ± SD of three independent experiments. # P<0.05 vs. control group; * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 vs. IL-1β group; ns, no significant difference (P>0.05). IL-1β, interleukin 1 β; RUT, rutaecarpine; MMP, matrix metalloproteinase; COX2, cyclooxygenase 2; SOX9, SRY-box transcription factor 9; PI3K, phosphoinositide-3-kinase; COL II, collagen type II; AGG, aggrecan.

Article Snippet: Subsequently, primary antibodies against COL II (1:200; cat. no. 28459-1-AP, Proteintech Group, Inc.) and p65 (1:400; cat. no. 8242, Cell Signaling Technology, Inc.) were used to incubate the cells at 4°C overnight.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Control