mouse monoclonal antibody (mab1177) against human pigment epithelial-derived factor (pedf  (R&D Systems)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Angiogenesis by HC?HA, a Complex of Hyaluronan and the Heavy Chain of Inter-?-Inhibitor, Purified from Human Amniotic Membrane

doi: 10.1167/iovs.10-5888

Figure Lengend Snippet: Detection of human angiogenic and antiangiogenic factors in the HC·HA complex by Western blot and protein arrays. (A) Western blot. 2 μg HA in AM extract, pooled fractions after F1 and F2, and HC·HA (each containing 2 μg HA) were loaded to each well and electrophoresed on 4% to 15% (wt/vol) gradient acrylamide gels under denaturing and reducing conditions. Proteins were transferred to the nitrocellulose membrane, and the membrane was incubated sequentially with PEDF antibodies and the respective secondary antibody. Detection was enhanced with reagent. (B) Protein arrays. 1.5 mL (25 μg/mL HA) AM extract or purified HC·HA complex was mixed with the detection antibodies, followed by incubation with the individual blocked human angiogenesis array membrane overnight at 4°C. After wash, each membrane was incubated with the secondary antibody, and signals were detected with chemiluminescent light exposed to x-ray films. Ovals labeled with numbers indicated candidate proteins detected in HC·HA. 1, 2, and 3: insulin-like growth factor binding protein-1, -2, and -3 (IGFBP-1,-2, and -3), respectively. 4: platelet factor 4 (PF4) or CXCL4. 5: TIMP-1. (C) Relative quantitation of candidate proteins in the HC·HA complex. The array data on developed x-ray film were quantitated by scanning the film on a transmission-mode scanner, and the array image file was analyzed by image analysis software (Image J). The relative protein amount in the HC·HA complex was compared with that in AM. The extract was set as 1.0.

Article Snippet: Reagents, Antibodies, and Assays Fibronectin (Fn), vitronectin (Vn), VEGF, a mouse monoclonal antibody (MAB1177) against human pigment epithelial-derived factor (PEDF), and human angiogenesis arrays (catalog no. ARY007) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Membrane, Incubation, Purification, Labeling, Binding Assay, Quantitation Assay, Transmission Assay, Software

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 1. The characterizations of PLGANPs. (A) Scanning electron micrograph (SEM) of pDNA-PLGANPs. (B) Size distribution of PLGANPs by volume (%). (C) Agarose gel electrophoresis was used to evaluate the integrity and purity of pAAV2-PEDF extracted from PLGANPs. They were pAAV2-PEDF extracted from PLGANPs (P-P) and untreated pAAV2-PEDF (U-P), respectively. (D) MTT assay was used to examine the cell cytotoxicity of bPLGANPs and PEI at different concentrations against CT26s, 48 h post-treatment (n=3). The data are expressed as mean ± SD.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: Agarose Gel Electrophoresis, MTT Assay

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 2. The effects of PEDF-PLGANPs in vitro. (A) The supernatants from CT26s of NS, AAV2-PLGANPs and PEDF-PLGANPs group were, respectively, detected by SDS-PAGE. PEDF protein (50 kDa) was only probed in CT26s treated with PEDF-PLGANPs. (B) Apoptotic CT26s of PEDF-PLGANPs, AAV2-PLGANPs and NS group were evaluated by flow cytometry with the values of 61.9, 15.2 and 13.2%, respectively. (C) The inhibitory effect of PEDF-PLGANPs on the proliferation of HUVECs was performed with a series of 1/2 dilutions from 1:2 to 1:64. The inhibitory rates of PEDF-PLGANP treated group declined with the increase of dilutions while AAV2-PLGANPs or NS group had no significantly change.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: In Vitro, SDS Page, Flow Cytometry

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 3. The study of PEDF-PLGANPs on the mice bearing CT26 tumors. (A) Tumor volumes of the mice bearing CT26 tumors in each group. (B) The tumors of experimental mice were weighed on the day 33 after CT26 inoculation when the mice were sacrificed. The values are expressed as mean ± SD. *P<0.05 compared with the tumor weight of AAV2-PLGANPs or NS group.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques:

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 4. The expression of PEDF gene loaded in PLGANPs in vivo. PEDF protein (50 kDa) could be only detected in CT26 tumors treated with PEDF-PLGANPs (P-P) while none was found in CT26 tumors from AAV2- PLGANPs (A-P) or NS treated mice.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: Expressing, In Vivo

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 5. Related factors to the growth inhibition of CT26 tumors. (A) Apoptotic cells within CT26 tumors were detected by TUNEL assay. Apoptotic CT26s (green) of NS, AAV2-PLGANPs and PEDF-PLGANPs group were, respectively, observed under a fluorescence microscope (original magnification, x200). (B) The apoptotic indexes (%) of CT26 tumors treated with NS, AAV2-PLGANPs and PEDF-PLGANPs. (C) CT26 tumors from NS, AAV2-PLGANPs and PEDF-PLGANP-treated mice were stained with H&E (original magnification, x400). (D) CD31 immunoreactive microvessels were seen from the sections of NS, AAV2-PLGANPs and PEDF-PLGANP-treated tumors. (E) The quantitative data manifested that MVD per 400 field of PEDF-PLGANP- treated tumors had a significant decrease compared with that of NS or AAV2-PLGANP-treated tumors. The data are expressed as mean ± SD. *P<0.05 compared with AAV2-PLGANPs or NS group.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: Inhibition, TUNEL Assay, Fluorescence, Microscopy, Staining