Anti-Fibronectin antibody

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody (AB2413)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling Fibronectin with ab2413 at 1/50 dilution. Antigen retrieval performed with EDTA buffer pH 8 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Fibronectin antibody (AB2413)

ICC/IF image of ab2413 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2413, 1 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in Hek293, HepG2 and MCF7 cells.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody (AB2413)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human kidney tissue, staining Fibronectin with ab2413.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibronectin antibody (AB2413)

ab2413 staining Fibronectin in human kidney tissue section by IHC-P (Formalin/PFA-fixed paraffin embedded tissue sections). Tissue sections were incubated with ab2413 at a dilution of 1 : 250 for one hour. Heat mediated antigen retrieval technique was used with citrate buffer at pH 6.0. DAB staining was done with a biotinylated secondary for 45 min at RT at a concentration of 1 : 1000.

• WB

Lab

Western blot - Anti-Fibronectin antibody (AB2413)

All lanes:

Western blot - Anti-Fibronectin antibody (ab2413) at 1 µg/mL

Lane 1:

Human colon tissue lysate at 10 µg

Lane 2:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 262 kDa

Observed band size: 285 kDa

false

• IHC

CiteAb

Immunohistochemistry - Anti-Fibronectin antibody (AB2413)

Immunohistochemistry-immunofluorescence using Anti-Fibronectin antibody, ab2413. Publication image from Farber, C. R. et al., 2016, Nat Commun, 27126736. Legend direct from paper.

Activation of RhoA promotes CTGF–VEGF complex formation in ECM, whereas inactivation of RhoA induces MMP3-mediated CTGF cleavage and VEGF release.(a–d) Immunofluorescence analysis of the binding of VEGF to ECM in different MSC cultures. MSCs were incubated with the indicated treatments for 7 days. Immunofluorescence staining was performed on non-permeabilized cells using antibodies against CTGF (a), fibronectin (b), collagen I (c) or VEGF (d). (e,f) MSCs were transfected with siRNA-control or siRNA-CTGF, then cultured with the indicated medium for 6 days. Western blot (WB) analysis of the cell lysates was performed using antibodies against CTGF and β-actin (e). Immunofluorescence staining was performed on non-permeabilized cells using antibodies against CTGF or VEGF (f). (g–j) WB and co-immunoprecipitation (IP) analyses of VEGF and CTGF in the overlay media of MSCs with different treatment. MSCs were incubated with the indicated treatments for 7 days. Overlay media : M1, M2, M3 and M4 were collected, respectively. Western blotting analysis of the overlay media was performed using antibodies against CTGF (g), MMP3 (h) and VEGF (i). Overlay media were subjected to IP assays using antibody against VEGF, the VEGF-associated CTGF was detected by western blotting with antibody against CTGF (j). (k,l) WB analysis of VEGF and CTGF in the ECMs of the cultured MSCs with different treatment. MSCs were incubated with the indicated treatment for 7 days. ECMs : E1, E2, E3 and E4 were collected. Western blotting analysis of the ECMs was performed using antibodies against CTGF (k) and VEGF (l). (m–o) WB and co-IP analysis of VEGF and CTGF in the overlay media of MSCs transfected with empty vector (EV) or L63RhoA. In the aliquots of the overlay medium collected from the cells overexpressed L63RhoA, 50 or 100 ng ml−1 rMMP3 was added and the reactions were maintained at 37 °C for 2 h. Overlay media were subjected to immunoprecipitation assays using antibody against VEGF, the VEGF-associated CTGF was detected by western blotting with antibody against CTGF (m). Western blotting analysis of the overlay media was performed using antibodies against CTGF (n) and MMP3 (o). Scale bars, 50 µm. Data are representative of three independent experiments.

• IHC

CiteAb

Immunohistochemistry - Anti-Fibronectin antibody (AB2413)

Immunohistochemistry-immunofluorescence using Anti-Fibronectin antibody, ab2413. Publication image from Farber, C. R. et al., 2016, Nat Commun, 27126736. Legend direct from paper.

Increased MMP3 in ECM and VEGF signalling in EYFP+ cells were seen at the intima layer of the injured arteries in mice treated with ROCK inhibitor.Nestin-Cre : : ROSA26-EYFP mice were subjected to either sham surgery or wire insertion-induced injury in femoral arteries 4 weeks (4W) after procedure. Fasudil at a dose of 30 mg kg−1 dissolved in water was administered by an oral gavage twice daily to mice, starting at 3 days before the surgery until 4 weeks after. Immunofluorescence staining of the artery sections using antibodies against CTGF, fibronectin and collagen I (a, in red). Double-immunofluorescence staining of the artery sections using antibodies against EYFP (green) and MMP3 (red) (b). *E, elastic fibre. White arrows represent double-positive staining. 4,6-Diamidino-2-phenylindole stains nuclei blue. Scale bars, 50 µm. Quantification of the number of MMP3+ cells per tissue area (N. MMP3+ cell per mm2; c). n=5. Data are represented as mean±s.e.m. *P<0.01 determined by ANOVA. Double-immunofluorescence staining of the artery sections using antibodies against EYFP (green) and p-VEGFR2 (red) (d). A, adventitia layer; I, intima layer; M, media smooth muscle layer; *E, elastic fibre. White arrows represent double-positive staining cells. DAPI stains nuclei blue. Scale bars, 50 µm. Quantification of the number of p-VEGFR2+ cells per tissue area (N. p-VEGFR2+ cell per mm2; e). n=5. Data are represented as mean±s.e.m. *P<0.001 versus Sham. #P<0.001 versus injury 4 W+Veh (vehicle) group as determined by ANOVA. (f) Schematic model indicating RhoA-MMP3-CTGF-VEGF axis in the fate decision of MSCs during arterial remodelling. Activation of RhoA stimulates CTGF expression and CTGF–VEGF complex formation in ECM, resulting in differentiation of MSCs into SMC/myofibroblasts (left panel); inactivation of RhoA promotes MMP3 production, which cleavages CTGF and releases VEGF from CTGF–VEGF complex, leading to endothelial differentiation of MSCs (right panel).

• WB

CiteAb

Western blot - Anti-Fibronectin antibody (AB2413)

Western Blotting using Anti-Fibronectin antibody, ab2413. Publication image from Arbonés-Mainar, J. M. et al., 2017, Redox Biol, 28600981. Legend direct from paper.

Linezolid effect on secreted fibronectin (FN1) and apolipoprotein E (APOE) levels. A) Protein loading control. Image of a SDS-PAGE gel for adipocyte-differentiated human adipose tissue-derived stem cells (hASCs) secretome samples after Coomassie blue staining. B) FN1 levels. Immunoblot image from adipocyte-differentiated hASCs exposed to different linezolid concentrations. C) Graph representing fibronectin relative quantity. D) APOE levels. Immunoblot image from adipocyte-differentiated hASCs exposed to different linezolid concentrations. As we were particularly interested in the APOE protein, and it had been reported that the specificity of many antibodies to its target protein is very low [63], we verified its specificity by performing peptide mass fingerprinting of the band obtained from the Western blot analysis of the culture medium. APOE was one of the two detected proteins in this band (Table S4). E). Graph representing APOE quantity. M, U, D, DL30 and DL60 code for molecular weight marker, undifferentiated hASCs, 0, 30 and 60 µM linezolid-exposed adipocyte-differentiated hASCs, respectively. Dashed line (100%) represents the mean values for linezolid-untreated adipocyte differentiated hASCs. The bars indicate the protein percentage of the undifferentiated and linezolid-treated adipocyte-differentiated hASCs. Error bars represent the standard deviation. *, P ≤ 0.0433 (versus linezolid-untreated adipocyte differentiated hASCs). #, P = 001 (versus 30 µM linezolid-treated adipocyte-differentiated hASCs).

false

• WB

CiteAb

Western blot - Anti-Fibronectin antibody (AB2413)

Western Blotting using Anti-Fibronectin antibody, ab2413. Publication image from Teupser, D. et al., 2016, Nat Commun, 27665711. Legend direct from paper.

Vigilin controls levels of secretory proatherogenic proteins.Secretome of primary hepatocytes isolated from 10-week-old mice injected with either Ad-shCtrl or Ad-shVig (n=8 per group; four biological replicates with each two technical replicates) was collected from the medium and quantified using label-free mass spectrometry (MS-LFQ). (a) Cumulative distribution function plot displaying fold-changes in secretion upon knockdown of vigilin in primary hepatocytes of top 100 PAR-CLIP targets (based on cumulative crosslinked reads, red line), poor targets (remaining targets, blue line) and non-targets (not crosslinked in both PAR-CLIP replicates, grey line). (b) Volcano plot of differentially secreted proteins upon vigilin knockdown in primary hepatocytes. x axis : Log2 fold-change of intensities, y axis : -Log10P values. Significant hits among secreted top 100 PAR-CLIP targets (based on T-to-C counts) are indicated in red dots, other significant targets in blue, non-targets and non-significant hits in grey. Significance was determined using false discovery rate (FDR)-corrected (FDR=0.01), permutation-based multiple t-tests (250 x) and curve bend s0=0.5. (c) Plot of differentially secreted PAR-CLIP targets (x axis) against T-to-C reads (y axis) indicates downregulation of more frequently bound targets. Significance was determined by Pearson's correlation test. (d) In vivo validation of MS-LFQ data through side-by-side immunoblot analysis of six targets from blood plasma of mice upon gain- (left panel : Ad-GFP and Ad-VIGILIN) and loss-of-function (Ad-shCtrl, Ad-shVig and PBS) from Fig. 2. (e) VIGILIN EMSAs representing binding sites on apoB and fetuin-A mRNAs identified by PAR-CLIP. Upper panel : alignment of vigilin PAR-CLIP sequence reads to gene loci of Apob and Ahsg (fetuin-A) mRNA CDS'. RREs are highlighted in yellow. The kernel density of T-to-C (T>C) transitions detected in PAR-CLIP reads is shown in red bars, the T-to-C conversion probability density of the cluster sequence is shown in blue bars. The read depth of the cluster is shown in grey. The percentage change of T-to-C transitions is indicated below the nucleotide sequence on a colour scale from blue to yellow. Lower panel : autoradiograph of EMSAs performed using binding site sequences identified by PAR-CLIP, mutated RREs (indicated in red) and scrambled sequences of these sites. The RNA sequences are indicated below.

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• WB

CiteAb

Western blot - Anti-Fibronectin antibody (AB2413)

Western Blotting using Anti-Fibronectin antibody, ab2413. Publication image from Teupser, D. et al., 2016, Nat Commun, 27665711. Legend direct from paper.

Knockdown of hepatic vigilin reduces atherosclerotic plaque formation.(a) Quantification of hepatic vigilin knockdown in male Ldlr−/− mice with weekly injections of two different GalNAc-conjugated siRNAs targeting vigilin (siVig-GalNAc#1 : n=10, siVig-GalNAc#2 : n=10) or PBS (n=9) for 18 weeks starting at 4 weeks of age. Values are shown relative to PBS-injected control mice. (b) Immunoblot analysis of vigilin targets from blood plasma. ApoM and apoA-I were used as loading controls. Time course of plasma (c) cholesterol and (d) triglyceride levels throughout treatment period. Fractionated blood plasma from treated mice indicating VLDL, LDL and HDL particles that were quantified through (e) cholesterol and (f) triglyceride levels in each fraction. Quantification of (g) plasma NEFA, (h) hepatic triglyceride and (i) cholesterol levels. (j) Quantification of plasma bile acid levels. (k,l) Characterization of atherosclerosis in mice from a. (k) Representative oil red O-stained aortic root sections and (l) quantification of the lesion areas. Scale bar, 200 µm. *P<0.05, **P<0.01 and #P<0.05, ##P<0.01, ###P<0.001 determined by ANOVA with Tukey's (in c and d) or Holm-Sidak (in a, g–j) post hoc analysis. All data are shown as the mean±s.d.

false