XB-IMG-130951
Xenbase Image ID: 130951
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FIG. 4. The XK81 gene is a direct target for XAP-2. (A) Fertilized
eggs were injected with a mixture of RNAs encoding chordin (500
pg) and glucocorticoid-inducible GRXAP-2 (1 ng). Ectodermal explants
were isolated at stage 7–8, and divided into four groups. Two
groups were treated with 10 g/ml cycloheximide (CHX ) to
inhibit protein synthesis. After 15 min, two groups (DEX ) were
treated with 10 M dexamethasone (DEX) to activate the accumulated
GRXAP-2 fusion protein. Controls were treated with an
equivalent volume of the DEX solvent (ethanol) alone. After
culturing to stage 12 equivalent, RNA was isolated and processed
for Northern blot analysis. (A) Hybridization to the XK81 keratin
probe revealed that the GRXAP-2 fusion protein responded as
expected to the DEX administration, resulting in partial recovery of
XK81 expression (DEX vs. DEX-), and that this was not prevented
by cycloheximide (CHX vs. CHX-). Note that different exposures
are shown for XK81 and EF1 probes. (B) Since cycloheximide
treatment has a general inhibitory effect on RNA synthesis in
Xenopus embryos, phosphorimage analysis was performed to normalize
the XK81 expression data to that of the control, EF1 , and
the results shown in histogram format. Based on this analysis,
induction of XK81 by XAP-2 appeared to have a similar magnitude
in the presence or absence of cycloheximide. (C, D) Loss of
competence during gastrulation. Fertilized eggs were injected with
a mixture of chordin and GRXAP-2 RNAs as in panel A. Ectodermal
explants were isolated at stage 7/8, and subsets transferred to
medium containing 10 m DEX at the stages indicated, from stage
8.5 through stage 13. Following culture to the equivalent of stage
20 (to minimize the temporal variable in DEX exposure), RNA was
isolated for Northern blot analysis. Hybridization with an XK81
keratin probe revealed strong recovery of keratin expression when
DEX was added immediately (stage 8.5; note that the uninjected
lane was loaded with a 1/10 dilution to facilitate exposure), but that
this response rapidly declined for subsequent DEX additions.
Hybridization with control EF1 probe showed essentially no
differences, and hybridization with a XAP-2 probe indicated that the injected GRXAP-2 RNA (endogenous XAP-2 hybridization not
visible at this exposure) was not differentially affected by the DEX
treatment. Phosphorimager quantification of Northern blot data
are shown in panel D. Image published in: Luo T et al. (2002) Copyright © 2002. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |