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Recombinant pdgf bb

Manufactured by R&D Systems
Sourced in United States

Recombinant PDGF-BB is a recombinant protein produced in E. coli cells. It is a homodimeric form of the PDGF-BB isoform, which is a member of the platelet-derived growth factor family. PDGF-BB is a signaling protein involved in cell growth and division.

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4 protocols using recombinant pdgf bb

1

Notch Signaling in Vascular Remodeling

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Recombinant Tat101 was purchased from ImmunoDiagnostics. Recombinant PDGF-BB was purchased from R&D Systems. Jagged-1 and DAPT were purchased from Sigma. Antibodies were obtained from the following sources: Ki67 (abcam), NICD (cell signaling), Actin (sigma), α-SM, Notch3 (cell signaling), VEGF-A (abcam); goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology. Flag-tagged CSL-VP16 plasmids were obtained from Dr. Aly Karsan (University of British Columbia, Vancouver, Canada) and control Notch3 siRNA (sc-29798), RBPJ siRNA (sc-41446), and scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology.
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2

Boyden Chamber Migration Assay for RPE Cells

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Migration was measured using a modified Boyden chamber assay, as previously described [51 (link)]. Briefly, 5 × 104 RPE cells that were treated in serum-free DMEM with 0, 0.05, 0.1, 0.3, 0.5 or 0.7 μM TSA for 24 h were seeded in the upper part of a Boyden chamber together with the corresponding concentrations of TSA in 24-well plates. Inserts were coated with fibronectin (2 μg/cm2). The lower chamber was filled with 0.4% FBS-DMEM containing 10 ng/mL of recombinant PDGF-BB (R&D Systems Inc., Minneapolis, MN). After 5 hours of incubation, the inserts were washed three times with PBS, fixed with cold methanol (4°C) for 10 minutes, and counterstained with hematoxylin for 20 minutes. The number of migrated cells was counted by phase-contrast microscopy (×320). Four randomly chosen fields were counted per insert.
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3

Cell Viability Assay and Signaling Pathway

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The EZ-Cytox Cell Viability Assay Kit were purchased from Daeil Lab Service (Seoul, Korea) and cell culture materials were purchased from Gibco-BRL (Gaithersburg, MD, USA), respectively. Recombinant PDGF-BB was obtained from R&D systems (Minneapolis, MN, USA). Specific antibodies for GAPDH, ERK1/2, phosphorylation of ERK1/2, p38, and phosphorylation of p38 for analysis of western blots, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA).
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4

Evaluating the Impact of ALK5 Silencing on Endothelial and Pericyte Migration

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Conditioned media from PCs pretreated with ALK5 siRNA or scrambled siRNA was collected at 72 hours post-transfection and applied to human brain microvascular ECs for 48 hours. ECs were trypsinized and immediately added to the top of Boyden chamber polycarbonate membranes (Corning Costar, 5 μm pores) precoated with 0.1% gelatin. The lower compartment of the Boyden chamber contained serum-free M199 with VEGF-A (100 ng/ml). ECs were allowed to migrate for 10 hours from the upper chamber towards the VEGF-A gradient (lower compartment). The membrane was immersed in 4% paraformaldehyde for 20 minutes, and then its upper surface was scraped with a cotton swab to remove non-migrated cells. Cells on the bottom surface (i.e., migrated cells) were imaged and counted after staining with 0.1% Crystal Violet. For PC migration, following treatment with ALK5 siRNA or scrambled siRNA, human brain PCs were trypsinized, resuspended in LG DMEM with TGFβ1 (5 ng/ml) and immediately added to the top of Boyden chamber polycarbonate membranes (Corning Costar, 8 μm pores) precoated with 0.1% gelatin. The lower compartment of the Boyden chamber contained serum-free LG DMEM with TGFβ1 (5 ng/ml) and recombinant PDGF-BB (R&D, 10 ng/ml). PCs were allowed to migrate for 6 hours. Membranes with migrated PCs were fixed, stained, imaged and quantified as described above.
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