XB-IMG-78195
Xenbase Image ID: 78195
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Fig. 3. Hypoxia increases the amount of 4E-BP protein which binds to eIF4E.
(A) Western blot analysis. Protein extracts from embryos exposed to 21%, 10% and 5% O2, at stages 21 and 25 were resolved by electrophoresis, blotted and reacted with anti-4E-BP antibodies (9452, Cell Signaling). Anti-α tubulin antibodies were used as a loading control.
(B) Densitometric quantification of 4E-BP from immunoblots in the different experimental conditions as described in material and method. *P<0.05; **P<0.005; ***P<0.001. (C) Isolation of eIF4E and its partners from stage 25 embryo extracts was performed using m7GTP Sepharose beads before Western blot analysis. Immunoblots with anti-4E-BP and
anti-eIF4E antibodies were performed. An anti-tubulin alpha antibody was used as a purification control in m7GTP samples. 4E-BP binds eIF4E. (D) qRT-PCR of 4E-BP mRNA
in normoxia (21% O2) and hypoxia (10 and 5% O2) at stages 21 and 25. No significant variation of the mRNA amount was detected. (E) Western blot analysis. Protein extracts from embryos exposed to several concentration of LY294002, a well-established inhibitor of PI3-kinase which acts upstream of Akt, were resolved by electrophoresis, blotted and reacted with anti-4E-BP and anti-Thr308-Akt antibodies. Anti-α tubulin antibodies were used as a loading
control. Treatment with 50 μM LY294002 affected Akt phosphorylation state and triggered an increase of 4E-BP protein amount. (F) Western blot analysis. Protein extracts from embryos exposed to rapamycin, a compound known to block mTOR activity, were resolved by electrophoresis, blotted and reacted with anti-4E-BP and anti-Thr308-Akt antibodies. Anti-α tubulin antibodies were used as a loading control. Treatment with rapamycin induced a dose-dependent increase of the 4E-BP amount. Image published in: Hidalgo M et al. (2012) Copyright © 2012. Image reproduced with permission of the Publisher, The Company of Biologists Ltd. Larger Image Printer Friendly View |