Hypoxia stimulates Src, PLD1, and PKCγ phosphorylation in a VEGF-dependent manner in retina. (A) C57BL/6 mice pups were exposed to 75% oxygen from P7 to P12, at which time they were returned to normoxia for the indicated time periods, enucleated, retinas were isolated, and analyzed by Western blotting for Src, PLD1, and PKCγ phosphorylation using their phosphospecific antibodies. (B) After exposure to hyperoxia, pups were returned to normoxia and administered 1 μg of Scr or VEGF siRNA at P12 and P13 by intravitreal injections. Retinas were isolated at P15, and the proteins were analyzed by Western blotting for Src, PLD1, and PKCγ phosphorylation using their phosphospecific antibodies. The blots were reprobed with antibodies of the indicated molecules for normalization. The blots in panels A and B that were probed with phosphospecific anti-PKCγ antibodies were also reprobed with either anti–β-tubulin or anti-VEGF antibodies for the purpose of lane loading control or to show the effect of VEGF siRNA on VEGF levels. (C) Retinas isolated at P15 were analyzed by double or triple immunofluorescence staining for phosphorylation of Src (C), PLD1 (D), and PKCγ (E) along with CD31 as described in “Methods.” The right column shows the higher magnification (original magnification ×40) of the selected areas of images shown in the left column.