XB-IMG-136024
Xenbase Image ID: 136024
|
Figure 2. Expression of synthetic mRNAs af.
ter oocyte injection. {A) Schematic representa·
tion of wild-type and mutated BMP and ac·
tivin molecules. The RXXR amino acid cleav·
age site sequences in the BMP and acti vin
molecules were mutated to GVDG in the
cleavage mutant constructs. Native molecules
form dimers {the disulfide bond is indicated),
which are then cleaved to form the carboxyterminal
mature ligand {shaded region) and
the amino-terminal precursor remainder pep·
tide (hatched region). The cleavage mutants do
not mature; thus, oocytes injected with the
cleavage mutant constructs only generate the
high molecular weight uncleaved polypeptide.
The relative positions of HA and Flag epitopes
within the pro-domain are indicated. (B) In
vivo translation of wild-type and cleavage mutant
mRNAs. Media from oocytes injected
with 50-200 ng of the indicated mRNAs were
pulse labeled (3 hrs) with (35S)methionine
and then analyzed by 14% SDS-PAGE. under
reducing conditions. Wild-type activin is
cleaved to form two polypeptides (37 and 14
kD) {asterisks, lane 21, whereas Cm- activin is
not {51 kD) (asterisk, lane 3). Both XBMP-7
and XBMP-4 mRNA·injected oocytes produced
cleaved amino-terminal peptides [34 and 35 kD, respectively) {asterisks, lanes 4,6). HA- Cm- XBMP-7 and Cm-XBMP-4 mRNA·
injected oocytes failed to generate the cleaved polypeptides and produced only the uncleaved form (S 1 and 48 kD, respectively)
(asterisks, lanes 5,7). Medium from HA- Cm- XBMP-7-injected oocytes was immunoprecipitated before SDS·PAGE {lane 5). Uninjected
oocytes are shown in lane 1. HA·tagged Cm- XBMP-7 was found to have activity similar to Cm- XBMP-7. (C) Maturation of FlagXBMP
· 7 and Flag- XBMP-4 proteins is inhibited by expression of Cm-XBMP-7. (Lane 1) Uninjected oocytes; (lane 2) HA- Cm-XBMP-7
mRNA {50 ng)·injected oocytes. {Lanes 3 and 4, respectively) Injection of Flag- XBMP-7 or Flag- XBMP-4 mRNA {SOng) results in the
formation of cleaved precursor remainder peptides (asterisk at 34 and 3S kD, respectively) in addition to uncleaved monomers
(arrowhead at 51 and 48 kD, respectively) and uncleaved dimers farrow at 102 and 96 kD, respectively). {Lanes 5 and 6, respectively)
Coinjection of Cm- XBMP-7 mRNA (200 ng) with Flag- XBMP-7 mRNA ISO ng) or Flag-XBMP-4 mRNA ISO ng) results in a decreased
amount of precursor remainder peptide. This indicates that processing of Flag-XBMP· 7 and Flag-XBMP-4 is inhibited in the presence of
Cm- XBMP-7. 1Lanes 7 and 8, respectively) Coinjection of Cm- activin mRNA (200 ng) with Flag-XBMP-7 mRNA (SOng) or Flag- XBMP-4
mRNA ISO ng) does not significantly reduce the formation of the precursor remainder peptide, indicating that the inhibition of processing
is specific to the BMPs. Flag-tagged XBMP-7 and XBMP-4 were found to have similar, although slightly weaker, activity compared to native
XBMP· 7 and XBMP-4. All samples are run under nonreducing conditions. All lanes are immunoprecipitated with either the HA flane 2 only)
or Flag antibodies. Image published in: Hawley SH et al. (1995) Copyright © 1995. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article. Larger Image Printer Friendly View |