Figure 2
Figure 2. VEGF-A protein expression is reduced in αB-crystallin−/− mice during OIR. In panels A through F, expression of VEGF-A (A,C,D,F; red) and relationship with isolectin-B4+ endothelial cells (B,C,E,F; green) in wild-type (A-C) and αB crystallin−/− mice (D-F) with OIR at P17. Bar indicates 50 μm. Original magnification, ×100. Images were obtained by the use of a SpotII digital camera and fluorescence microscope (Leica) with a 40×/0.75 NA lens. In panel G, VEGF-A protein concentration (pg/mg protein) in posterior eyecup homogenates, determined by ELISA in αB crystallin−/− and wild-type mice with OIR. αB crystallin−/− mice at P17 showed significantly lower concentrations of VEGF-A than wild-type (P < .001) mice. In panel H, VEGF-A mRNA expression determined by real-time PCR analysis and normalized to beta-actin mRNA in posterior eye cups of αB crystallin−/− and wild-type mice with OIR (n = 6). VEGF-A mRNA is up-regulated at P17 in wild-type mice (P < .005) and αB crystallin−/− mice (P < .02). In panel I, Hif-1α protein expression in posterior eyecups with or without hyperoxia at P12 and P17 as measured by Western blot. Hif-1α expression is up-regulated at P17 in wild-type and αB−/− mice. In panel J, αB-crystallin and its phosphorylated serine 59 form in wild-type murine retinas at P12 with or without hyperoxia, as measured by Western blot. Expression of phosphorylated serine 59 form of αB-crystallin is significantly increased in the presence of hyperoxia (n = 3; P < .01). In panel K, Western blot for αB-crystallin, VEGF, and VEGF-R2 in anti–VEGF-A immunoprecipitates from wild-type and αB crystallin−/− mice at P12 and P17 in OIR. αB-crystallin binds to VEGF-A at P12 and P17 in wild-type mice but not in αB crystallin−/− mice. Knockout of αB-crystallin does not affect VEGF-R2 binding to VEGF-A. In panel L, Western blot for αB-crystallin and TGF-β in anti–TGF-β immunoprecipitates of wild-type and αB crystallin−/− mice at P12 and P17 in OIR. There is no apparent binding of αB-crystallin to TGF-β at P12 and P17 in αB crystallin−/− and wild-type mice.

VEGF-A protein expression is reduced in αB-crystallin−/−mice during OIR. In panels A through F, expression of VEGF-A (A,C,D,F; red) and relationship with isolectin-B4+ endothelial cells (B,C,E,F; green) in wild-type (A-C) and αB crystallin−/− mice (D-F) with OIR at P17. Bar indicates 50 μm. Original magnification, ×100. Images were obtained by the use of a SpotII digital camera and fluorescence microscope (Leica) with a 40×/0.75 NA lens. In panel G, VEGF-A protein concentration (pg/mg protein) in posterior eyecup homogenates, determined by ELISA in αB crystallin−/− and wild-type mice with OIR. αB crystallin−/− mice at P17 showed significantly lower concentrations of VEGF-A than wild-type (P < .001) mice. In panel H, VEGF-A mRNA expression determined by real-time PCR analysis and normalized to beta-actin mRNA in posterior eye cups of αB crystallin−/− and wild-type mice with OIR (n = 6). VEGF-A mRNA is up-regulated at P17 in wild-type mice (P < .005) and αB crystallin−/− mice (P < .02). In panel I, Hif-1α protein expression in posterior eyecups with or without hyperoxia at P12 and P17 as measured by Western blot. Hif-1α expression is up-regulated at P17 in wild-type and αB−/− mice. In panel J, αB-crystallin and its phosphorylated serine 59 form in wild-type murine retinas at P12 with or without hyperoxia, as measured by Western blot. Expression of phosphorylated serine 59 form of αB-crystallin is significantly increased in the presence of hyperoxia (n = 3; P < .01). In panel K, Western blot for αB-crystallin, VEGF, and VEGF-R2 in anti–VEGF-A immunoprecipitates from wild-type and αB crystallin−/− mice at P12 and P17 in OIR. αB-crystallin binds to VEGF-A at P12 and P17 in wild-type mice but not in αB crystallin−/− mice. Knockout of αB-crystallin does not affect VEGF-R2 binding to VEGF-A. In panel L, Western blot for αB-crystallin and TGF-β in anti–TGF-β immunoprecipitates of wild-type and αB crystallin−/− mice at P12 and P17 in OIR. There is no apparent binding of αB-crystallin to TGF-β at P12 and P17 in αB crystallin−/− and wild-type mice.

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