rabbit polyclonal antibody against total fn  (Abcam)

Journal: Journal of cellular biochemistry

Article Title: The Novel mTOR Complex 1/2 Inhibitor P529 Inhibits Human Lung Myofibroblast Differentiation

doi: 10.1002/jcb.25878

Figure Lengend Snippet: A) HLF were treated with 1 ng/ml TGF-β for 24 h and the indicated concentrations of P529. Lysates were subjected to western blotting for α-SMA, collagen-1, fibronectin, and GAPDH. N=3 biologic repeats. B) Densitometry of Col1/GAPDH and α-SMA/GAPDH. Measurements are normalized to the TGF-β treatment condition. Statistical significance was tested using student’s t-test. *, # denote statistically significant differences (p<0.05) between expression of α-SMA (*) or Col1 (#) in the TGF-β and TGF-β/P529 treatment conditions. C) Real time PCR of the indicated genes. Statistical significance was tested using student’s t-test. *, # denote statistically significant differences (p<0.05) between expression of α-SMA (*) or Col1 (#) in the TGF-β and TGF-β/P529 treatment conditions. N, denotes not statistically significant (FN and PAI-1).

Article Snippet: Rabbit polyclonal antibody against total fibronectin (FN) was from Abcam (ab2413, Cambridge, MA).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of cellular biochemistry

Article Title: The Novel mTOR Complex 1/2 Inhibitor P529 Inhibits Human Lung Myofibroblast Differentiation

doi: 10.1002/jcb.25878

Figure Lengend Snippet: HLF were treated with 1 ng/ml TGF-β for 24 h and P529 or vehicle control, as indicated. A) Cells were then subjected to staining with phalloidin (red) and DAPI (Blue) and visualized with a 60x objective. Scale bar = 50 μm. B) Cells were stained with anti-fibronectin antibody (green) and DAPI (Blue) and visualized with a 10x objective (top row) or a 60x objective (bottom row). Scale bar = 250 μm. C, D) HLF were transfected with siRNA targeting mTOR or scrambled control, followed by treatment with 1 ng/ml TGF-β for 24 h, followed by western blotting and densitometry of the indicated proteins. Statistical significance was tested using student’s t-test. * denote statistically significant differences (p<0.05) between conditions. E) HLF were plated and grown overnight on tissue culture plastic. Serum-free media with DMSO or 10 μM P529 was then added to induce growth arrest. Linear wounds were made at the time of serum removal and imaged. Cells were fixed after 24 h and imaged with a 10x objective. Images shown have been processed with ImageJ to enhance edges and contrast for clarity. F) Closure after 24 h was measured using Image J. Scale Bar = 200 μM. Statistical significance was tested using student’s t-test. * denote statistically significant differences (p<0.05) between conditions.

Article Snippet: Rabbit polyclonal antibody against total fibronectin (FN) was from Abcam (ab2413, Cambridge, MA).

Techniques: Control, Staining, Transfection, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Plasma fibronectin stabilizes Borrelia burgdorferi –endothelial interactions under vascular shear stress by a catch-bond mechanism

doi: 10.1073/pnas.1615007114

Figure Lengend Snippet: Dependence of B. burgdorferi–endothelial interactions under physiological shear stress on pFn. (A) Schematic illustrating initiation steps (tethering, dragging) of the B. burgdorferi–endothelial interaction cascade leading to bacterial transmigration across endothelial barriers into extravascular tissues. Tethering bacteria anchor to endothelial surfaces via tethers, pause repeatedly as they move over endothelial surfaces, but move faster than 100 μm⋅s−1. Dragging bacteria move more slowly (<100 μm⋅s−1) and are untethered. Both tethering and dragging are Fn-dependent in mouse PCVs (18). There are reduced numbers of B. burgdorferi tethering and dragging on primary human endothelial monolayers in flow chambers at typical PCV shear stress (1 dyn/cm2) following treatment with polyclonal anti-Fn antiserum (B) and depletion of pFn from serum in bacterial cultivation medium (C). Numbers of tethering and dragging GFP-expressing B. burgdorferi (strain GCB966) in the presence of nonspecific IgGs or polyclonal αFn IgGs were measured by manual counting. In B, GCB966 was cultivated in the presence of mouse blood before imaging. In C, bacteria were cultivated without mouse blood to eliminate all sources of pFn. In C, −pFn indicates pFn-depleted growth conditions; for +pFn samples, bacteria grown under pFn-depleted conditions were supplemented with human pFn (+pFn) to the concentration present in blood (0.3 mg/mL) before imaging. Summary values: mean ± SEM. Statistics: two-way ANOVA, Holm–Sidak posttest (n = 3 independent bacterial and endothelial cultures per group). *P < 0.05 vs. IgG (B) or −pFn (C) within the same interaction type.

Article Snippet: Monolayers were incubated with primary antibodies against polyclonal human Fn (rabbit, 1:400 dilution, catalog no. F3648; Sigma–Aldrich), monoclonal mouse vimentin (mouse, 1:200 dilution, catalog no. 0725; Dako Canada ULC), and monoclonal mouse tubulin (mouse, 1:500 dilution, catalog no. 9026; Sigma–Aldrich), followed by three 10-min washes with PBS/0.02% TX-100 (permeabilized cells) or PBS alone (nonpermeabilized cells).

Techniques: Transmigration Assay, Expressing, Imaging, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Plasma fibronectin stabilizes Borrelia burgdorferi –endothelial interactions under vascular shear stress by a catch-bond mechanism

doi: 10.1073/pnas.1615007114

Figure Lengend Snippet: Antibodies and peptides used in experiments

Article Snippet: Monolayers were incubated with primary antibodies against polyclonal human Fn (rabbit, 1:400 dilution, catalog no. F3648; Sigma–Aldrich), monoclonal mouse vimentin (mouse, 1:200 dilution, catalog no. 0725; Dako Canada ULC), and monoclonal mouse tubulin (mouse, 1:500 dilution, catalog no. 9026; Sigma–Aldrich), followed by three 10-min washes with PBS/0.02% TX-100 (permeabilized cells) or PBS alone (nonpermeabilized cells).

Techniques:

Journal: Acta biomaterialia

Article Title: Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications.

doi: 10.1016/j.actbio.2014.10.035

Figure Lengend Snippet: Fig. 3. (A) Schematic of the microcontact printing procedure, including free collagen hydrogel carboxyl group activation, ‘‘ink’’ printing and passivation steps. Fluorescence microscopical images of the resulting patterned (B) 30 lm and (C) 200 lm stripes on the surface of RHCIII–MPC hydrogels, visualized by staining with an anti-fibronectin antibody.

Article Snippet: The presence of FN in the micropatterns was confirmed by using a primary rabbit polyclonal antibody against FN (Abcam, Cambridge, UK), followed by a secondary antibody, donkey antirabbit IgG, conjugated with quantum dots Qdot655 (H+L) (Life Technologies, USA).

Techniques: Activation Assay, Fluorescence, Staining