Anti-Fibroblast activation protein, alpha 抗体

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fibroblast activation protein, alpha antibody (AB53066)

Immunohistochemistry analysis of paraffin-embedded human breast carcinoma tissue sections labeling Fibroblast activation protein, alpha with ab53066 (without peptide-right and with peptide- left).

• WB

Unknown

Western blot - Anti-Fibroblast activation protein, alpha antibody (AB53066)

Blocking buffer : 5% (w/v) non-fat dry milk in TBST.

Primary antibody dilution buffer : 5%（w/v）non-fat dried milk，0.1%（v/v）, Tween-20 in TBST.

Secondary antibody dilution buffer : 5%（w/v）non-fat dried milk，0.1%（v/v），Tween-20 in TBST.

12% SDS gel. Nitrocellulose membrane.

Blocking : Room temperature for 2 hours or overnight at 4°C. Then wash 3x for 5 minutes with 0.05% blocking buffer.

Primary antibody incubation : diluted in TBST at 1/500. Incubate overnight with 4 degrees shaking. Then, in 0.05% TBST, wash membrane 3-4 times for 10min.

Secondary antibody incubation : diluted in TBST at 1/2000. Incubate 37°C for 1 hour. Then, in 0.05% TBST, wash membrane 3-4 times for 10min.

ECL development.

All lanes:

Western blot - Anti-Fibroblast activation protein, alpha antibody (ab53066) at 1/500 dilution

Lane 1:

LOVO cell extract

Lane 2:

LOVO cell extract at 40 µg with immunising peptide

Predicted band size: 88 kDa

Observed band size: 90 kDa

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• WB

CiteAb

Western blot - Anti-Fibroblast activation protein, alpha antibody (AB53066)

Fibroblast activation protein, alpha western blot using anti-Fibroblast activation protein, alpha antibody ab53066. Publication image and figure legend from Evrard, S. M., Lecce, L., et al., 2016, Nat Commun, PubMed 27340017.

ab53066 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab53066 please see the product overview.

Oxidative stress promotes EndMT and has additive effects to TGF-β.(a) Representative dihydroethidium (DHE) microscopy images of thoracic aortic sections from tamoxifen-induced end.SclCreERT;R26RstopYfp;ApoE-/- mice after 8 versus 18 weeks HFD demonstrating in situ superoxide production. Analysis by one-way ANOVA; overall p<0.0001. (b) TUNEL assay on atherosclerotic lesions from tamoxifen-induced end.SclCreERT;R26RstopYfp;ApoE-/- mice after 8 versus 18 weeks HFD demonstrating apoptotic Yfp+ cells. For a and b : L=lumen; scale bars, 100 μm. Insets at higher magnification as indicated. Arrows indicate co-positive cells. Staining performed on at least three spatially separated thoracic aortic sections per mouse (n=5 mice per group). Control experiments from non-atherosclerotic mice shown in Supplementary Fig. 8. (c) Phase contrast microscopy of HUVECs grown in basal media (ctrl) or following exposure to TGF-β and/or H2O2 for 5 days. Scale bars, 100 μm. (d) Heatmap showing expression of endothelial genes (top), non-endothelial genes also downregulated in EndMT/EMT, genes known to be upregulated in EndMT/EMT and mesenchymal genes (bottom) for HUVECs exposed to control conditions (basal media—unstimulated), TGF-β or TGF-β plus 200 μM H2O2. Because there are no gene sets for 'EndMT' or 'fibroblast' in databases like GO, KEGG, Reactome or MSigDB, this gene list was compiled by extensive literature search (Supplementary Table 2). (e) FAP protein expression by western blot with quantification in HUVECs exposed to control (ctrl) conditions (unstimulated) or TGF-β, with and without H2O2 at 100 or 200 μM. FAP ladder (L) represents 100 kDa (upper) and 75 kDa (lower intense band). FAP expected molecular weight/size is 90 kDa. GAPDH ladder represents 37 kDa. GAPDH expected molecular weight/size is 37 kDa. (f) FAP RNA levels assessed by qRT-PCR under identical conditions. (g) CD31 protein by western blot with quantification. CD31 ladder (L) represents 150 kDa (upper) and 100 kDa (lower intense band). CD31 expected molecular weight/size is 130 kDa. GAPDH ladder represents 37 kDa. GAPDH expected molecular weight/size is 37 kDa. (h) CD31 RNA levels assessed by qRT-PCR is presented for HUVECs under identical conditions. Data in e–h analysed by two-way ANOVA with complete results in Supplementary Table 3. (i) Cell migration for unstimulated HUVECs versus after treatment with TGF-β only or TGF-β+200 μM H2O2 for 5 days. Analysis by one-way ANOVA; overall P=0.015. (j) Cell invasion for unstimulated HUVECs versus after treatment with TGF-β only or TGF-β+200 μM H2O2 for 5 days. Analysis by one-way ANOVA; overall p<0.0001. n=6–9 for qRT-PCR and 3–6 for western blots. Migration and invasion determined from three independent experiments with each experiment having n=3. NS, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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