Journal: Development (Cambridge, England)

Article Title: Developmental vascular pruning in neonatal mouse retinas is programmed by the astrocytic oxygen-sensing mechanism

doi: 10.1242/dev.175117

Figure Lengend Snippet: PHD2 deficiency promotes HIF-α accumulation and VEGF-A expression in retinal astrocytes. (A) Quantitative RT-PCR (qPCR) analysis for PHD2, VEGF-A and EPO. Total RNA used for qPCR was isolated from primary retinal astrocytes cultured from neonatal mice (P3). All data points were normalized against β-actin values. (B) ELISA for VEGF-A and EPO. Conditioned media from retinal astrocyte cultures were collected and assayed with VEGF and EPO ELISA kits. (C) Western blotting of nuclear extracts isolated from cultured retinal astrocytes. For quantification, PHD2, HIF-1α and HIF-2α signals were normalized to that of α-actin. n=5 mice/group. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: For ELISA, conditioned media were collected from retinal astrocyte cultures, and used for VEGF-A and EPO ELISA kits, respectively (R&D Systems, now part of Thermo Fisher; catalog numbers MMV00 for VEGF kit, and MEP00B for EPO kit).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot