primary antibodies against pedf  (GeneTex)

Journal: Breast Cancer Research : BCR

Article Title: Loss of pigment epithelium-derived factor: a novel mechanism for the development of endocrine resistance in breast cancer

doi: 10.1186/bcr3356

Figure Lengend Snippet: Pigment epithelium-derived factor expression in human breast cancer cell lines . (a) Pigment epithelium-derived factor (PEDF) and estrogen receptor alpha (ERα) protein levels were detected by western blot analysis in several human breast cancer cell lines, including hormone-dependent T47D, MCF-7, and ZR-75-1 cells, tamoxifen-resistant BT474 cells, tamoxifen-resistant and aromatase inhibitor-resistant MCF-7:5C and MCF-7:2A cells, and ER-negative MDA-MB-231 cells. (b) PEDF mRNA expression level in the different breast cancer cell lines was determined by quantitative real-time PCR analysis with PUM1 used as a normalization control. (c) Conditioned media were analyzed by western blot for the presence of PEDF protein in the indicated breast cancer cell lines. Staining of proteins by Coomassie blue included as a loading control. (d) Hormonal regulation of PEDF and ERα protein in MCF-7 and T47D breast cancer cells. Cells were incubated in phenol red-free RPMI media supplemented with 10% charcoal-stripped FBS for 72 hours and, subsequently, were treated with vehicle, 1 nM 17β-estradiol (E2), or 1 nM E2 + 1 μM 4-hydroxytamoxifen (4OHT) for 24 hours, and the PEDF and ERα protein level was determined by western blot analysis. β-actin was used as a normalization control. All experiments were performed in triplicate independently.

Article Snippet: Membranes were probed with primary antibodies against PEDF (Chemicon Inc., Temecula, CA., USA), against ERα and phospho-Ser167-ERα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), against RET, p-RET (Y1062), mammalian target of rapamycin (mTOR), p-mTOR and AKT, and against pAKT, MAPK, pMAPK and p70S6K (Cell Signaling Technology Inc., Danvers, MA, USA), and against β-actin (Sigma Chemical Co., St Louis, MO, USA).

Techniques: Derivative Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Staining, Incubation

Journal: Breast Cancer Research : BCR

Article Title: Loss of pigment epithelium-derived factor: a novel mechanism for the development of endocrine resistance in breast cancer

doi: 10.1186/bcr3356

Figure Lengend Snippet: Pigment epithelium-derived factor expression in primary and recurrence breast tumor tissues . (a) Immunohistochemistry (IHC) staining for pigment epithelium-derived factor (PEDF) and estrogen receptor alpha (ERα) were performed on tissue microarrays generated from normal breast tissue (left panel), primary breast tumor tissue ( n = 59; middle panel), and recurrence breast tumor tissue ( n = 59; right panel). Cores used to generate tissue microarrays (TMAs) were 0.6 mm in diameter. Tumor tissues were also stained in the absence of a PEDF antibody to act as a negative control (left panel, inset). PEDF staining was quantified as an intensity score ranging from 0 to 255. A scale of 0 to 3 was used to score staining intensity of ERα. (b) Western blot analysis of normal tissue (N), primary breast tumor tissue (PT), or recurrence tumor tissue (RT) to assess PEDF, total ERα, and phosphorylated ERα (Ser118 and Ser167) protein level. β-actin was used as a loading control. (c) Representative quantitative real-time PCR analysis of PEDF and ER α mRNA expression in N, PT, or RT. For experiment, total RNA was extracted from paraffin-embedded tissues using Trizol and analyzed by real-time PCR as described in Materials and methods. PEDF and ER α mRNA were normalized to the internal control gene PUM1 . All experiments were performed in triplicate. PEDF mRNA level was statistically significantly lower in the recurrence tumor tissue compared with the primary tumor tissue. * P < 0.001.

Article Snippet: Membranes were probed with primary antibodies against PEDF (Chemicon Inc., Temecula, CA., USA), against ERα and phospho-Ser167-ERα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), against RET, p-RET (Y1062), mammalian target of rapamycin (mTOR), p-mTOR and AKT, and against pAKT, MAPK, pMAPK and p70S6K (Cell Signaling Technology Inc., Danvers, MA, USA), and against β-actin (Sigma Chemical Co., St Louis, MO, USA).

Techniques: Derivative Assay, Expressing, Immunohistochemistry, Generated, Staining, Negative Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Loss of pigment epithelium-derived factor: a novel mechanism for the development of endocrine resistance in breast cancer

doi: 10.1186/bcr3356

Figure Lengend Snippet: Effect of recombinant pigment epithelium-derived factor on the estrogen receptor alpha signaling pathway . (a) Western blot analysis of pigment epithelium-derived factor (PEDF), estrogen receptor alpha (ERα), phospho-ER, phospho-Akt, mitogen-activated protein kinase (MAPK), phospho-MAPK, rearranged during transfection (RET), pRET, p70S6K, and mammalian target of rapamycin (mTOR) protein expression in MCF-7, MCF-7:5C, and 5C-PEDF cells. (b) Effect of recombinant PEDF on ERα, pERα, RET, and pRET (Y1062) protein expression in MCF-7:5C cells. Cells were treated with 100 nM recombinant PEDF (rPEDF) protein for 24 hours and cell lysates were analyzed by western blot. β-actin was used as a loading control. (c) Effect of rPEDF on estrogen response element (ERE) luciferase activity in MCF-7 and MCF-7:5C cells. MCF-7 and MCF-7:5C cells were grown in estrogen-free RPMI media and then co-transfected with a 5× ERE luciferase plasmid and a renilla reporter plasmid for 24 hours. Following transfection, cells were treated with 1 nM 17β-estradiol (E2), 100 nM rPEDF, or E2 + rPEDF for 24 hours and luciferase activity was measured. Values presented as relative luciferase activity after normalization to Renilla luciferase activity. Data expressed as mean ± standard deviation of the results obtained from triplicate experiments. Basal ERE activity was statistically significantly higher in MCF-7:5C cells compared with MCF-7 cells. * P < 0.01.

Article Snippet: Membranes were probed with primary antibodies against PEDF (Chemicon Inc., Temecula, CA., USA), against ERα and phospho-Ser167-ERα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), against RET, p-RET (Y1062), mammalian target of rapamycin (mTOR), p-mTOR and AKT, and against pAKT, MAPK, pMAPK and p70S6K (Cell Signaling Technology Inc., Danvers, MA, USA), and against β-actin (Sigma Chemical Co., St Louis, MO, USA).

Techniques: Recombinant, Derivative Assay, Western Blot, Transfection, Expressing, Luciferase, Activity Assay, Plasmid Preparation, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Loss of pigment epithelium-derived factor: a novel mechanism for the development of endocrine resistance in breast cancer

doi: 10.1186/bcr3356

Figure Lengend Snippet: Effect of recombinant pigment epithelium-derived factor (rPEDF) on the growth of endocrine-sensitive MCF-7 and endocrine-resistant MCF-7:5C breast cancer cells in vitro and in vivo . (a) MCF-7 and MCF-7:5C cells were treated with increasing concentrations of rPEDF, rPEDF + anti-PEDF antibody (anti-PEDF), or anti-PEDF antibody for 7 days, and cell proliferation was determined using a DNA quantitation kit as described in Materials and methods. Experiments were repeated three times, and data are shown as mean ± standard deviation (SD). * P < 0.001 compared with untreated controls. (b) MCF-7:5C cells were treated with rPEDF, anti-rPEDF, or rPEDF + anti-rPEDF for 72 hours and apoptosis was determined by TUNEL staining. Bar graph: summary of percentage of apoptotic cells counted in five fields from three experiments. Data presented as mean ± SD. (c) Effect of rPEDF on the growth of MCF-7:5C cells in vivo . MCF-7:5C cells were bilaterally injected into the mammary fat pad of ovariectomized nude mice, and when tumors reached an area of 0.1 cm 2 the mice ( n = 15/group) were randomized into two treatment groups: PBS or rPEDF. The mean cross-sectional tumor area was measured up to 30 days. Bar graph: mean cross-sectional tumor area in the control group and the PEDF-treated group. (d) Intratumoral microvessel density (MVD) in tumor tissues was determined by immunohistochemical staining by an endothelial-specific antibody CD34; PBS group (×200) and PEDF group (×200). Quantitative analysis of microvessel density is also shown. Data presented as mean ± SD.

Article Snippet: Membranes were probed with primary antibodies against PEDF (Chemicon Inc., Temecula, CA., USA), against ERα and phospho-Ser167-ERα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), against RET, p-RET (Y1062), mammalian target of rapamycin (mTOR), p-mTOR and AKT, and against pAKT, MAPK, pMAPK and p70S6K (Cell Signaling Technology Inc., Danvers, MA, USA), and against β-actin (Sigma Chemical Co., St Louis, MO, USA).

Techniques: Recombinant, Derivative Assay, In Vitro, In Vivo, Quantitation Assay, Standard Deviation, TUNEL Assay, Staining, Injection, Immunohistochemical staining

Journal: Investigative ophthalmology & visual science

Article Title: Propranolol Attenuates Proangiogenic Activity of Mononuclear Phagocytes: Implication in Choroidal Neovascularization.

doi: 10.1167/iovs.18-25502

Figure Lengend Snippet: FIGURE 4. Propranolol (Pro) increases PEDF expression in J774 macrophages leading to antiangiogenic effect on choroidal explant. PEDF and TSP-1 present in J774 CM following 24-hours stimulation with propranolol was assessed using dot blot (A) and ELISA-based assay (B) (N ¼ 3, *P < 0.05 versus CTL). (C) Representative images of J774 cells incubated with or without propranolol (10 lM) immunostained with anti-PEDF antibody (green) and DAPI (blue). Scale bar: 40 lm. (D) Western blot analysis for PEDF and beta-actin expression in J774 cells incubated with or without propranolol (10 lM). (E) Endothelial cells sprouting from choroidal explant immunostained with anti-PEDF receptor antibodies (green), lectin (red), and DAPI (blue), scale bar: 30 lm. (F) Representative images of endothelial cells sprouting from choroidal explant incubated for 24 hours with recombinant PEDF (1, 1.5, and 2 ng/mL) added to CM from J774; or incubated for 24 hours with CM from J774. Basal DMEM was used for CTL media. Quantification of vascular area was performed with ImageJ software and are presented in histogram (N ¼ 4–5, **P < 0.01 versus CTL media, ##P < 0.01 versus J774 CM). (G) Representative images of endothelial cells sprouting from choroidal explant incubated for 24 hours with basal DMEM (CTL media), CM from J774 incubated with or without propranolol (10 lM), and with or without anti-PEDF antibody. Quantification of vascular area was performed with ImageJ software and are presented in histogram (N ¼ 3–5, *P < 0.05, **P < 0.01 versus CTL media).

Article Snippet: After blocking, the membranes were probed with specific primary antibodies against PEDF (1:300; sc-25994; Santa Cruz Biotechnology), b1AR, b2-AR, b3-AR (1:400 sc-568, sc-9042, sc-50436, respectively; Santa Cruz Biotechnology), and b-actin (1:400; sc-47778; Santa Cruz Biotechnology).

Techniques: Expressing, Dot Blot, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Recombinant, Software

Journal: Investigative ophthalmology & visual science

Article Title: Propranolol Attenuates Proangiogenic Activity of Mononuclear Phagocytes: Implication in Choroidal Neovascularization.

doi: 10.1167/iovs.18-25502

Figure Lengend Snippet: FIGURE 5. PEDF and PEDF-R expression increase following propranolol treatment and are associated with caspase-3 cleaved in choroid in mouse laser-induced CNV model. (A) Representative images of cryosection of choroid immunostained with anti-PEDF receptor antibody (green), F-actin (red), and DAPI (blue). Scale bar: 20 lm. (B–F) QPCR and immunofluorescence analysis were performed respectively on choroid and retina section from mice at D14 after receiving or not laser burn and treated with or without propranolol (6 mg/kg/d). (B) QPCR analysis for PEDF-receptor and PEDF mRNA expression from RPE/choroid complex (N ¼ 3, *P < 0.05 versus CTL). (C) PEDF-R/PEDF mRNA expression ratio (N ¼ 3, *P < 0.05 versus CTL). (D) Confocal imaging of retinal cryosection immunostained with anti-PEDF antibody (red), anti-CD11b antibody (white), and DAPI (blue). Magnification of white square focused on CD11b-positive cell closed to CNV. Green arrow shows PEDF expression inside CD11b-positive cell. Scale bar: 20 lm. (E) Confocal imaging of retinal cryosection immunostained with anti–cleaved caspase-3 antibody (red), TRITC-lectin (red), and DAPI (blue). Magnification of white square focused on colocalization of cleaved caspase-3 in choroid, scale bar: 20 lm. (F) QPCR analysis for caspase-3 mRNA expression from RPE/choroid complex (N ¼ 3, *P < 0.05, **P < 0.01 versus CTL).

Article Snippet: After blocking, the membranes were probed with specific primary antibodies against PEDF (1:300; sc-25994; Santa Cruz Biotechnology), b1AR, b2-AR, b3-AR (1:400 sc-568, sc-9042, sc-50436, respectively; Santa Cruz Biotechnology), and b-actin (1:400; sc-47778; Santa Cruz Biotechnology).

Techniques: Expressing, Imaging