goat anti serpin f1 pedf  (R&D Systems)

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA relative to β-actin reference gene in retinal cells freshly isolated and after 3d exposure to NBA, siCNL, 50 nM siCASP2 and PEDF/PEDF-34/PEDF-44. b % of surviving βIII-tubulin + RGC after 3d in culture in NBA, and after treatment with siCNL, 50 nM siCASP2 and PEDF/PEDF-34/PEDF-44. c Fold change in CASP2 mRNA relative to β-actin reference gene in retinal cells freshly isolated and after 3d exposure to suboptimal doses of siCASP2 (5–20 nM) with and without PEDF/PEDF-34/PEDF-44. d % of surviving βIII-tubulin + RGC after 3d in culture in with suboptimal concentrations of siCASP2 (5–20 nM) with and without PEDF/PEDF-34/PEDF-44 ( n = 9 wells/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Isolation

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA at 7d after ONC + PBS, ONC + siCNL and ONC + siCASP2 and ONC + PEDF treatment. b, c Western blot and subsequent densitometry to show that ONC-induced C-CASP2 (p12 fragment) was significantly suppressed by siCASP2 and PEDF treatment ( n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA after ONC + saline and ONC + PEDF-34 daily eye-drops over 21d. b , c Western blot and subsequent densitometry to show that ONC-induced rise in C-CASP2 (p12 fragment) is significantly suppressed at 1d and 3d after injury and treatment, without changes in C-CASP3 or C-CASP6 levels. d Immunohistochemistry to show that ONC-induced localisation of C-CASP2 (red) in βIII-tubulin + (green) RGC (arrowheads) after ONC + saline treatment is suppressed by 1d and 3d after ONC + PEDF-34 eye-drops. Scale bar = 50 µm. GCL = ganglion cell layer. (n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Saline, Eye Drops, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA in 5-FDU-treated cultures showing no change in CASP2 mRNA after siCASP2 or PEDF treatment compared to cultures with glia. b % of surviving βIII-tubulin + RGC in 5-FDU treated cultures in siCASP2 treated cells was unaffected but survival was reduced in the absence of glia. c Fold change in CASP2 mRNA in purified RGC cultures showing no change in CASP2 mRNA after siCASP2 or PEDF treatment. d % of surviving βIII-tubulin + RGC in purified RGC cultures in siCASP2 treated cells was unaffected but survival was reduced in the absence of glia ( n = 9 wells/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Purification

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a ELISA to determine levels of BDNF, CNTF, GDNF, NGF and NT-3 released into the culture medium. Treatment of retinal cultures with ELISA isolated levels of BDNF, GDNF and NGF or CNTF either alone or in combination partially suppresses b CASP2 mRNA and C-CASP2 protein c , d by western blot and subsequent densitometry. e Representative images to demonstrate morphology of βIII-tubulin + RGC (note: neurite outgrowth occurs in surviving βIII-tubulin + RGC in wells containing PEDF, BDNF/GDNF/NGF and CNTF). (n = 9 wells/treatment). Scale bar in f = 20 µm

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Western Blot

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a – c Pre-treatment of PEDF and PEDF-34 with a polyclonal antibody to PEDF fails to reduce CASP2 mRNA ( a ) and protein levels ( b ) whilst PEDF is suppressed in the absence of PEDF polyclonal antibody treatment, as confirmed by densitometry ( c ). d Pre-treatment with PEDF polyclonal antibody also suppresses the RGC neuroprotective effects of PEDF and PEDF-34. (n = 9 wells/treatment). e Representative images of the treatments in d to show morphology of βIII-tubulin + RGC. Scale bar in e = 20 µm

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques:

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a CASP2-dependent apoptosis requires the formation of the PIDDosome (Adapted from ). b PEDF treatment suppresses CASP2, RAIDD but not PIDD and downstream Bim mRNA. c , d PEDF treatment suppresses C-CASP2, RAIDD, phopho-c-Jun and downstream Bim as detected by western blot and densitometry. e , f Immunohistochemistry to show changes in Bim protein colocalised to caspase-2 in the ganglion cell layer (GCL), inner plexiform layer (IPL), occasional cells of the inner nuclear layer (INL) whilst high power views of the GCL showed that Bim and caspase-2 were colocalised to RGC. Scale bars in e and ( f ) = 50 µm ( n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Western Blot, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

doi: 10.1111/j.1582-4934.2009.00731.x

Figure Lengend Snippet: PEDF expression in human cardiac tissue. PEDF and HIF-1α determination by Western blot of homogenized cardiac tissue isolated from two healthy hearts (lanes 1 and 2) and four explanted hearts from patients suffering from ischemic (lanes 3 and 4) and dilatative (lanes 5 and 6) cardiomyopathy (A). PEDF mRNA expression in human cardiac tissue isolated from the same hearts used for Western blot in (A), e.g. two healthy hearts (lanes 1 and 2) and four explanted hearts from patients suffering from ischemic (lanes 3 and 4) and dilatative (lanes 5 and 6) cardiomyopathy; GAPDH served as a loading control (B). Immunohistochemical staining of PEDF, troponin I and actin in paraffin embedded heart tissue from a healthy heart and explanted hearts from patients suffering from ischemic and dilatative cardiomyopathy, respectively (C). mRNA was isolated from the left ventricle of healthy human hearts ( n = 4) and from hearts of patients suffering from ischemic ( n = 8; * P = 0.014) or dilatative cardiomyopathy ( n = 17; n.s., P = 0.287); real-time PCR was performed employing specific primers for PEDF. Values represent mean values ± S.D. Values are given as x -fold of control and were normalized using GAPDH levels (D).

Article Snippet: We used an anti-human PEDF goat antibody (15 μg/ml; R&D Systems) for PEDF staining, an anti-human cardiac troponin I rabbit monoclonal antibody (1/250 dilution; Abcam, Cambridge, UK) for cardiac muscle staining and an anti-human smooth muscle α-actin mouse monoclonal antibody (ready to use, BioGenex).

Techniques: Expressing, Western Blot, Isolation, Control, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Cytoplasmic Expression and Extracellular Deposition of an Antiangiogenic Factor, Pigment Epithelium-Derived Factor, in Human Atherosclerotic Plaques

doi: 10.1161/01.atv.0000175759.78338.1e

Figure Lengend Snippet: Figure 1. Detection of PEDF mRNA and protein in human aortic samples. a, Examples of RT-PCR analysis of PEDF mRNA (600 bp) in atherosclerotic lesions (A) and grossly nonatherosclerotic lesions (histological DIT, N). -actin (254 bp) was subsequently amplified as an internal control. RT-PCR was simultaneously performed without RT as a negative control (RT). b, Examples of the Western blot analysis of PEDF protein, corresponding to each of the samples shown in (a). Recombinant PEDF protein secreted into the culture medium of COS7 cells stably trans- fected a simian lentiviral vector expressing human PEDF (PEDF- CM) used as a positive control. Figure 2. Immunohistochemical detection of PEDF protein. a, Immunohistochemistry (IHC) for PEDF (red) in the human retina, used as a positive control. The bottom 2 panels are high- powered view of the boxed area shown in the corresponding upper panel, respectively. An intense reaction (red) for PEDF was seen in the inner/outer segments in the acellular zone con- taining rods and cones (anti-PEDF). No positive reaction was seen in the serial sections reacted with isotype-matched nonim- mune antibody (murine IgG1, upper left), as well as with primary antibody absorbed by excess recombinant PEDF (right upper and bottom). Original magnification: upper panels, 70; and the lower panels, 140. INL indicates inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. All sections were counterstained with hematoxylin. b, Immunohistochemical detection of PEDF in an aortic sample with an atherosclerotic lesion (case 20). Three typical lesions, namely fibrous cap (upper right), lipid core (lower left), and shoulder (lower right) lesions, are shown in high-powered views of the respective boxed areas in the upper left panel. A cytoplasmic staining pat- tern was observed in the fibrous cap and shoulder lesions, whereas a diffuse deposition pattern was observed in the necrotic lipid core. Original magnification: upper left, 20; others, 300.

Article Snippet: Immunohistochemistry was performed using 4% paraformaldehydefixed, paraffin-embedded tissue with the following antibodies, according to the standard streptavidin-biotin complex technique: goat anti-human PEDF antibody (15 g/mL) (R&D systems, Minneapolis, Minn), anti-CD68 (1:100; DAKO, Glostrup, Denmark), antiCD34 (1:100; Novocastra, Newcastle, UK), and anti–smooth muscle cell actin (HHF35) (1:100; Enzo Life Science, New York, NY).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Negative Control, Western Blot, Recombinant, Stable Transfection, Plasmid Preparation, Expressing, Positive Control, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Cytoplasmic Expression and Extracellular Deposition of an Antiangiogenic Factor, Pigment Epithelium-Derived Factor, in Human Atherosclerotic Plaques

doi: 10.1161/01.atv.0000175759.78338.1e

Figure Lengend Snippet: Figure 3. Double IHC to detect sources of PEDF-expressing cells in human aorta tissue containing atherosclerotic lesions. a and b, The 2 panels on the right are high-powered views of the boxed areas shown in the 2 respective panels on the left. The majority of the PEDF-expressing cells (red) were positive for CD68-positive monocytes/macrophages (a, right, brown, arrows), and some smooth muscle cells were positive for HHF35 (b, right, brown, arrows). Original magnification: left pan- els, 50; right panels, 250.

Article Snippet: Immunohistochemistry was performed using 4% paraformaldehydefixed, paraffin-embedded tissue with the following antibodies, according to the standard streptavidin-biotin complex technique: goat anti-human PEDF antibody (15 g/mL) (R&D systems, Minneapolis, Minn), anti-CD68 (1:100; DAKO, Glostrup, Denmark), antiCD34 (1:100; Novocastra, Newcastle, UK), and anti–smooth muscle cell actin (HHF35) (1:100; Enzo Life Science, New York, NY).

Techniques: Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Cytoplasmic Expression and Extracellular Deposition of an Antiangiogenic Factor, Pigment Epithelium-Derived Factor, in Human Atherosclerotic Plaques

doi: 10.1161/01.atv.0000175759.78338.1e

Figure Lengend Snippet: Figure 4. Relationship between intimal neovessels and the cytoplasmic PEDF staining pattern in human coronary arteries. a, Typical and representative findings of double IHC for the PEDF cytoplasmic staining pattern, which was seen in cell-rich areas. The upper and middle panels on the right show a high-powered view of the boxed area shown in the panel on the left, respectively. Upper 2 panels demonstrate double IHC labeling granular cytoplasmic staining for PEDF (red), where some neovessels were intermingled (brown, arrows). Middle 2 panels show double IHC with anti-PEDF (red) and anti-CD68 antibody (blue), indicating that PEDF-positive foam cells were proved to be also positive for CD68. Bottom 2 panels show double IHC using anti-CD34 (red) and anti-ssDNA (brown) antibody. Majority of neovessels (arrows) were negative for ssDNA (brown nuclei). b, Scattered plot analysis assessing the relationship between the number of PEDF-positive cells and that of CD34-positive vessels. Among all tissue sections, 67 cell-rich areas demonstrating an accumulation of cytoplasmically PEDF-positive cells were observed. The number of PEDF-positive cells was highly correlated to that of CD34-positive vessels (Spearman’s rank correlation, 0.686, P0.0001).

Article Snippet: Immunohistochemistry was performed using 4% paraformaldehydefixed, paraffin-embedded tissue with the following antibodies, according to the standard streptavidin-biotin complex technique: goat anti-human PEDF antibody (15 g/mL) (R&D systems, Minneapolis, Minn), anti-CD68 (1:100; DAKO, Glostrup, Denmark), antiCD34 (1:100; Novocastra, Newcastle, UK), and anti–smooth muscle cell actin (HHF35) (1:100; Enzo Life Science, New York, NY).

Techniques: Staining, Labeling