mouse monoclonal antibodies against human α sma  (R&D Systems)

Journal: Journal of Allergy

Article Title: Lithium Attenuates TGF- β 1 -Induced Fibroblasts to Myofibroblasts Transition in Bronchial Fibroblasts Derived from Asthmatic Patients

doi: 10.1155/2012/206109

Figure Lengend Snippet: Phenotypic shifts in nonasthmatic and asthmatic HBF populations induced by TGF- β 1 . Administration of TGF- β 1 resulted in a prominent FMT in AS HBF populations, illustrated by relatively high fractions of α -SMA-positive cells (A). This effect is independent of TGF- β RII levels (B) but correlates with nuclear accumulation of p-Smad2 (C). (A) HBFs from NA ( n = 8) and AS ( n = 8) were cultured in DMEM with 10% FCS for 24 hours, and then in serum-free conditions with or without 5 ng/mL TGF- β 1 for 7 days, and fractions of α -SMA-positive cells were detected. Each column represents the percentage of myofibroblasts for each individual HBF culture (±SE). In the AS group, the percentage of positive cells was significantly higher than in the NA group in TGF- β 1 -treated cultures (Kruskal-Wallis nonparametric ANOVA, * P < 0.05). (B) For the estimation of TGF- β RII levels, NA and AS HBFs were cultured in DMEM with 10% FCS for 24 hours, immunostained for TGF- β RII, and analyzed by both fluorescence microscopy and FACS. Representative fluorescence microscopy images (bar = 50 μ m) illustrate a similar pattern of expression of TGF- β RII in HBF from NA (a) and AS (b) cultures. Representative histogram showing the specificity of TGF- β RII staining in comparison with isotype control (c). TGF- β RII expression levels were similar in both studied groups. Each point of the graph (d) represents data obtained from a single cell population (NA or AS) and horizontal bars represent the mean values. (C) TGF- β 1 -induced p-Smad2 translocation to the nucleus in HBFs. Fibroblasts from NA and AS were cultured in serum-free conditions for 24 hours and then with (+TGF- β ) or without (control) TGF- β 1 (5 ng/mL), next cells were immunostained for p-Smad2 and photographed with fluorescence microscopy. After 30 minutes of treatment with TGF- β 1 , nuclear accumulation of p-Smad2 was detectable, and was more pronounced in AS HBFs (arrows). Bar = 50 μ m.

Article Snippet: In brief, cells growing on glass coverslips were fixed in 3.7% paraformaldehyde, permeabilised in 0.1% Triton X-100, blocked with 1% BSA, and incubated with a mouse monoclonal antibody against human α -SMA (clone 1A4, Sigma-Aldrich) and Alexa Fluor 488 goat anti-mouse IgG (clone A11001, Sigma-Aldrich).

Techniques: Cell Culture, Fluorescence, Microscopy, Expressing, Staining, Translocation Assay

Journal: Journal of Allergy

Article Title: Lithium Attenuates TGF- β 1 -Induced Fibroblasts to Myofibroblasts Transition in Bronchial Fibroblasts Derived from Asthmatic Patients

doi: 10.1155/2012/206109

Figure Lengend Snippet: The effect of LiCl on TGF- β 1 -stimulated FMT in non-asthmatic and asthmatic HBFs. HBFs from NA ( n = 11) and AS ( n = 10) groups were cultured in DMEM supplemented with 0.1% BSA and TGF- β 1 (5 ng/mL), LiCl (10 mM), or TGF- β 1 /LiCl (A, B). (A) Representative images of HBF from NA and AS culture: Nomarski interference contrast microscopic images (a–d), immunofluorescent staining of α -SMA (e–h), and Hoechst staining of cell nuclei (i–l). Bar = 50 μ m. (B) Graphs showing the percentage of myofibroblasts after 7-day culture of HBFs. Each point represents a single HBF culture derived from NA and AS groups (the means of at least three separate experiments for each culture are presented, in the one experimental point at least 100 cells were counted; only HBFs with α -SMA incorporated into stress fibres were considered as α -SMA positive, i.e, myofibroblasts). Bars on graphs represent mean values. The significance of differences between TGF- β 1 -stimulated HBFs without LiCl and with LiCl was determined by applying Kruskal-Wallis non-parametric ANOVA, * P < 0.05.

Article Snippet: In brief, cells growing on glass coverslips were fixed in 3.7% paraformaldehyde, permeabilised in 0.1% Triton X-100, blocked with 1% BSA, and incubated with a mouse monoclonal antibody against human α -SMA (clone 1A4, Sigma-Aldrich) and Alexa Fluor 488 goat anti-mouse IgG (clone A11001, Sigma-Aldrich).

Techniques: Cell Culture, Staining, Derivative Assay

Journal: Journal of Allergy

Article Title: Lithium Attenuates TGF- β 1 -Induced Fibroblasts to Myofibroblasts Transition in Bronchial Fibroblasts Derived from Asthmatic Patients

doi: 10.1155/2012/206109

Figure Lengend Snippet: The effect of inhibition of GSK-3 β on TGF- β 1 -stimulated FMT in nonasthmatic and asthmatic HBFs. HBFs from non-asthmatics ( n = 8) and asthmatics ( n = 8) were cultured in DMEM supplemented with 0.1% BSA and TGF- β 1 (5 ng/mL), TWS119 (10 μ M), or TGF- β 1 /TWS119 (A, B). (A) Representative images of HBF from NA and AS culture: Nomarski interference contrast microscopic images (a–d), immunofluorescent staining of α -SMA (e–h), and Hoechst staining of cell nuclei (i–l). Bar = 50 μ m. (B) Graphs showing the percentage of myofibroblasts after 7-day culture of HBFs. Each point represents a single HBF culture from NA and AS groups (the means of at least three separate experiments for each culture are presented, in the one experimental point, at least 100 cells were counted). Bars on graphs represent mean values. The significance of differences between TGF- β 1 -stimulated HBFs without TWS119 and with the inhibitor was determined by applying Kruskal-Wallis non-parametric ANOVA, * P < 0.05.

Article Snippet: In brief, cells growing on glass coverslips were fixed in 3.7% paraformaldehyde, permeabilised in 0.1% Triton X-100, blocked with 1% BSA, and incubated with a mouse monoclonal antibody against human α -SMA (clone 1A4, Sigma-Aldrich) and Alexa Fluor 488 goat anti-mouse IgG (clone A11001, Sigma-Aldrich).

Techniques: Inhibition, Cell Culture, Staining

Journal: Journal of Allergy

Article Title: Lithium Attenuates TGF- β 1 -Induced Fibroblasts to Myofibroblasts Transition in Bronchial Fibroblasts Derived from Asthmatic Patients

doi: 10.1155/2012/206109

Figure Lengend Snippet: The effect of inhibition of GSK-3 β on TGF- β 1 -stimulated α -SMA levels in nonasthmatic and asthmatic HBFs. HBFs from NA and AS groups were cultured for 7 days in DMEM supplemented with 0.1% BSA (control), or with the same medium with addition of TGF- β 1 (5 ng/mL), LiCl (10 mM), or TGF- β 1 /LiCl (a) or with the same control medium supplemented with TGF- β 1 (5 ng/mL), TWS119 (10 μ M) or TGF- β 1 /TWS119 (b). Protein extracts (10 μ g of protein per lane) were subjected to SDS-PAGE electrophoresis, and α -SMA protein/ α -tubulin (a) or α -SMA protein/GAPDH (b) was detected by immunoblotting. Responses were quantified by densitometry and normalized to the expression of α -tubulin or GAPDH, respectively. Data represents relative optical density mean of at least 3 experiments (ROD ± SE). Representative immunoblots are shown under the graphs.

Article Snippet: In brief, cells growing on glass coverslips were fixed in 3.7% paraformaldehyde, permeabilised in 0.1% Triton X-100, blocked with 1% BSA, and incubated with a mouse monoclonal antibody against human α -SMA (clone 1A4, Sigma-Aldrich) and Alexa Fluor 488 goat anti-mouse IgG (clone A11001, Sigma-Aldrich).

Techniques: Inhibition, Cell Culture, SDS Page, Electrophoresis, Western Blot, Expressing

Journal: BMC Veterinary Research

Article Title: Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts

doi: 10.1186/1746-6148-10-119

Figure Lengend Snippet: Sera effects on β-actin and α-SMA expression. Western blots were performed on whole cell lysate from EBF cultured for 4 days in DMEM containing 10% FBS or HS. Densitometry of β-actin and α-SMA was calculated by SynGene. (A) EBF cultured in HS show a higher α-SMA expression than EBF cultured in FBS (n = 5). Serum batch of (B) FBS and (C) HS (n = 2) did not affect α-SMA expression. F1 and F2 represent FBS batches, and H1 and H2 the HS batches. Data are presented as means ± SEM. (*p < 0.01).

Article Snippet: The membrane was then blocked in 3% BSA (PAA Laboratories) with TBST (20 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.05% [v/v] Tween-20) for 1 h at room temperature. α-SMA was detected following an overnight incubation of samples with mouse monoclonal antibody against human anti-α-SMA (1:1000; Sigma-Aldrich) and β-actin was detected using mouse monoclonal antibody against human anti-β-actin (1:10,000; Sigma-Aldrich) in 3% BSA-TBST at 4°C.

Techniques: Expressing, Western Blot, Cell Culture

Journal: PLoS ONE

Article Title: S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland

doi: 10.1371/journal.pone.0163981

Figure Lengend Snippet: Using sagittal sections of embryonic pituitaries on E18.5, staining for vascular endothelial cells with isolectin B4 ( Aʹ ) or α-SMA ( Dʹ ) was performed by visualization with Cy5 ( white ), together with staining of GFP (FITC; green , A and B ) and the pericyte marker DESMIN or PRRX1 (Cy3; red , Aʹʹ and Bʹʹ ). GFP/DESMIN-double ( yellow open arrowheads ), DESMIN/isolectin B4-double ( white open arrowheads ), GFP-single ( yellow arrows ), and DESMIN- or α-SMA-single ( white arrowheads ) positive cells are indicated. Merged images ( Aʹʹʹ , Bʹʹʹ , Dʹʹʹ , and Eʹʹʹ ) are shown on the right. Each cell type (n = 2 with two slices each) was counted, and results are listed in C and E . AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm ( Aʹʹʹ and Dʹʹʹ ) and 10 μm ( Bʹʹʹ and Eʹʹʹ ).

Article Snippet: The primary antibodies used were chicken immunoglobulin (Ig) Y against GFP (1:250 dilution, Aves Labs, Inc., Tigard, OR, USA), rabbit IgG against cow S100β (1:1000 dilution, Dako, Glostrup, Denmark), rabbit antiserum against rat PRRX1 (1:1,000 dilution, raised in our laboratory and assessed for specificity) [ ], rabbit antiserum against rat PRRX2 (1:1,000 dilution, raised in our laboratory and assessed for specificity) [ ], goat IgG against stem/progenitor cell marker human SOX2 (1:500 dilution, Neuromics, Edina, MN, USA), mouse monoclonal antibody against neural crest cell marker rat p75 (1:100 dilution, Abcom, Plc., Cambridge, UK), mouse monoclonal antibody against smooth muscle cell marker human α-SMA (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse antiserum against pericyte and a neural and mesenchymal stem/progenitor cell marker rat NESTIN (1:250 dilution, BD Biosciences, San Jose, CA, USA), mouse monoclonal antibody against mesenchymal cell marker pig VIMENTIN (1:10,000 dilution, Sigma-Aldrich Corp., St. Louis, MO, USA) and rabbit antibody against dividing cell marker human Ki67 (1:500 dilution, Abcom, Plc.).

Techniques: Staining, Marker

Journal: Oncotarget

Article Title: Resveratrol suppresses myofibroblast activity of human buccal mucosal fibroblasts through the epigenetic inhibition of ZEB1 expression

doi: 10.18632/oncotarget.7763

Figure Lengend Snippet: The expression of ZEB1 in BMFs could induce the formation of stress fibers which comprised of α-SMA (green lines) and lead to the transdifferentiation into myofibroblasts to secret collagen and cause tissue contraction of oral cavity. Treatment of resveratrol could upregulate the expression of miR-200c and EZH2/H3K27me3 to repress ZEB1 expression and leads to the inhibition of myofibroblast properties in fibrotic BMFs and may benefit to OSF disease, the pre-cancerous lesion of oral cavity.

Article Snippet: Monoclonal mouse anti-human antibody against α-SMA (1A4) and rabbit polyclonal anti-human antibody against COL1A1 (H-197) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Inhibition