Both Kdr and Flt1 mediate hypoxia-induced Src-PLD1-PKCγ-cPLA₂ activation in the mouse retina. (A,B) C57BL/6 mice pups were exposed to 75% oxygen from P7 to P12, at which time they were returned to normoxia to develop relative hypoxia. At the indicated time periods of hypoxia, their eyes were enucleated and retinas isolated, and tissue extracts were prepared. An equal amount of protein from normoxic and various periods of hypoxic retinal extracts was immunoprecipitated with anti-Kdr or anti-Flt1 antibodies, and the immunocomplexes were analyzed by western blotting using anti-PY20 antibodies. The blots were reprobed with anti-Kdr or anti-Flt1 antibodies for normalization. (C) After exposure to hyperoxia, mice pups were returned to normoxia and administered 1 μg/0.5 μL control, Kdr, or Flt1 siRNA at P12 and P13 by intravitreal injections. At P15, retinas were isolated and extracts prepared and analyzed by western blotting for pSrc, pPLD1, pPKCγ, and pcPLA₂ levels using their phosphospecific antibodies. The blots were reprobed with anti-Src, anti-PLD1, anti-PKCγ, anti-cPLA₂, anti-Flt1, anti-Kdr, or anti-β-tubulin antibodies for normalization or to show the effect of siRNAs on their target molecules. (D) All the conditions were the same as in panel C except that the retinal extracts were analyzed for cPLA₂ activity. The bar graphs represent the quantitative analysis of 3 independent experiments. The values are presented as mean ± SD. *P < .01 vs normoxia or control siRNA; ^†P < .05 vs control siRNA + hypoxia; ^§P < .01 vs control siRNA + hypoxia.