Hypoxia-induced Src-PLD1-PKCγ-cPLA₂ activation requires the production of both VEGFA and VEGFB. (A) An equal amount of protein from normoxic or various time periods of hypoxic retinal extracts was analyzed by western blotting for VEGFA and VEGFB levels using their respective antibodies. The blot was reprobed with anti-β-tubulin antibodies for normalization. (B) All the conditions were the same as in Figure 4C except that the mice pups were administered with control, VEGFA, or VEGFB siRNA at P10 and P11 by intravitreal injections, and at P12 the pups were returned to normoxia. After 12 hours the retinas were isolated, extracts prepared, and an equal amount of protein from normoxic and hypoxic retinas was immunoprecipitated with anti-PY20 antibodies; the immunocomplexes were analyzed by western blotting for Flt1 and Kdr using their specific antibodies. An equal amount of protein from normoxic and hypoxic retinas was also analyzed by western blotting for Flt1, Kdr, VEGFA, and VEGFB levels using their specific antibodies. (C) All the conditions were the same as in Figure 4C except that VEGFA or VEGFB siRNAs were administered at P12 and P13 and retinal extracts were analyzed for phosphorylation of Src, PLD1, PKCγ, and cPLA₂ using their phosphospecific antibodies. The blots were reprobed with anti-Src, anti-PLD1, anti-PKCγ, anti-cPLA₂, anti-VEGFA, anti-VEGFB, or anti-β-tubulin antibodies either for normalization or to show the downregulation of VEGFA and VEGFB by their respective siRNAs. The bar graphs in panels A-C represent the quantitative analysis of 3 independent experiments. The values are presented as mean ± SD. *P < .01 vs normoxia + control siRNA; ^†P < .05 vs hypoxia + control siRNA; ^§P < .01 vs hypoxia + control siRNA.