rhplgf  (R&D Systems)

Journal: The FASEB Journal

Article Title: Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems

doi: 10.1096/fj.201901353R

Figure Lengend Snippet: Recombinant PlGF has specific affinity with its mAb (PL5D11D4) and disrupts HREC barrier function. A, B) Dot immunoassay demonstrated the specific affinity of PlGF antibody (PL5D11D4) with rbPlGF (A) and rhPlGF (B). rhVEGF and BSA acted as negative controls. HRP, horseradish peroxidase. C) HRECs were cultured and grown with endothelial growth medium 2 (EGM-2) (with growth factors such as VEGF-A). After the cells grew to confluence, the culture medium was changed to endothelial cell growth basal medium 2 (EBM-2) (without growth factors), and PBS control and rhPlGF protein (100 ng/ml) were added to the culture medium and incubated for at least 2 d. TEER was monitored by an ECIS system at an AC frequency of 4 KHz in real time. TEER curves of HRECs that were treated with rhPlGF protein and PBS control. Error bars represent sd out of 4 duplicate samples. The experiments were repeated at least 3 times. D) WB result for VE-cadherin, β-catenin, and ZO-1. β-Actin was used as the protein loading control. E, F) Immunofluorescence staining results of tight junction ZO-1 and adhesion protein VE-cadherin. ****P < 0.0001.

Article Snippet: Dot immunoblotting assay Modification of the Dot immunoblotting assay ( 23 , 24 ) was used to validate the affinity and specificity of PL5DLLD4 antibody with human PlGF. rhPlGF protein (200 ng; 264-PGB-010/CF; R&D Systems), VEGF-165 (200 ng, 293-VE-010/CF; R&D Systems), and 500 ng bovine serum albumin (BSA) (MilliporeSigma) were deposited in the volume of 2 μl on dry nitrocellulose membrane (Bio-Rad) and immobilized by 15 min incubation at room temperature The membranes were blocked with 5% nonfat milk (Bio-Rad) in PBS at room temperature for 1 h and then incubated overnight at 4°C with mouse PL5DLLD4 antibody 0.5 μg/ml.

Techniques: Recombinant, Cell Culture, Control, Incubation, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems

doi: 10.1096/fj.201901353R

Figure Lengend Snippet: PlGF regulates G6PD, PRDX6, and EC barrier function through VEGFR1 and VEGFR2 signaling. A) WB results for G6PD. HRECs that were treated with rhPlGF and VEGF-165 protein (100 ng/ml each, for 2 d) were used for the WB assay: β-actin was used as the protein loading control. Note that rhPlGF, but not VEGF-165, down-regulated the protein level of G6PD compared with the PBS control. B) Double immunofluorescence labeling of G6PD and PRDX6. The HRECs that were treated with the PBS control, rhVEGF(a), and rhPlGF protein were fixed and stained with G6PD (green) and PRDX6 (red). The DAPI staining of the nucleus (blue) was used as the counterstaining. Note that the 2 proteins were colocalized in the PBS-treated and rhVEGF-treated cells (yellow); however, the staining signals were reduced in the rhPlGF-treated cells. The complete images of all 3 fluorescent staining channels are shown in Supplemental Fig. S5. C) WB results of ZO-1, VE-cadherin, and β-catenin. The confluent HRECs were treated with PBS, PBS + rhPlGF (100 ng/ml), rhPlGF + VEGFR1 antibodies (100 µg/ml), rhPlGF + VEGFR2 antibody (100 µg/ml), and VEGFR1 antibody alone or VEGFR2 antibody alone for 2 d. Note that rhPlGF protein down-regulated the protein levels of ZO-1, VE-cadherin, and G6PD, which were prevented by the VEGFR1 or VEGFR2 antibody. The antibody alone did not change the protein levels, which were equivalent to those of the PBS control. D) WB results of ZO-1, VE-cadherin, β-catenin, occludin-1, claudin-5, and G6PD. The confluent HRECs were treated with the PBS, PlGF/VEGF heterodimer (50, 100, or 200 ng/ml), PlGF (100 ng/ml), or VEGF-165 (100 ng/ml). Note that PlGF, the PlGF/VEGF heterodimer (200 ng/ml), and VEGF-165 down-regulated ZO-1, VE-cadherin, β-catenin, and claudin-5 (but not occludin-1); however, only PlGF and PlGF/VEGF down-regulated G6PD. E) TEER (or resistance) of HRECs. After the HRECs grew to confluence, the PBS control, PlGF/VEGF, or VEGF-165 was added to the medium, and the TEER was measured with ECIS in real time. The TEER values of the PlGF/VEGF heterodimer (blue) and VEGF-165 (purple) were significantly lower than those of the PBS control (red). Error bars represent sd out of 4 duplicate samples. F) WB results of PlGF/VEGF heterodimers in HREC lysate and culture medium. The confluent HRECs were treated with PBS, 25 mM d-glucose [high glucose (HG)], and 25 mM l-glucose [normal glucose (NG)]. Note that compared with the PBS controls, the HG up-regulated PlGF/VEGF in the cell lysates but decreased the levels in the culture medium. The β-actin was presented in the HREC lysate (but not in the culture medium) and used as the protein loading control. ****P < 0.0001.

Article Snippet: Dot immunoblotting assay Modification of the Dot immunoblotting assay ( 23 , 24 ) was used to validate the affinity and specificity of PL5DLLD4 antibody with human PlGF. rhPlGF protein (200 ng; 264-PGB-010/CF; R&D Systems), VEGF-165 (200 ng, 293-VE-010/CF; R&D Systems), and 500 ng bovine serum albumin (BSA) (MilliporeSigma) were deposited in the volume of 2 μl on dry nitrocellulose membrane (Bio-Rad) and immobilized by 15 min incubation at room temperature The membranes were blocked with 5% nonfat milk (Bio-Rad) in PBS at room temperature for 1 h and then incubated overnight at 4°C with mouse PL5DLLD4 antibody 0.5 μg/ml.

Techniques: Control, Immunofluorescence, Labeling, Staining