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Using antibodies against homeoprotein XIHbox 1 from Xenopus laevis, we have detected a new embryonic protein with a much larger molecular weight, 115 kDa. Antibodies fractionated according to their affinity for 3 different domains of the XIHbox 1 protein were used to show that this new protein is related to the C-terminal region of XIHbox 1 protein, downstream from the homeodomain. By immunohistochemistry, the protein was shown to be localized in nuclei of embryonic cells. On SDS-polyacrylamide gels, the 115 kDa protein appears as a set of closely spaced bands whose pattern varies with the stage of development and with the parental origin of the embryos. The protein could be extracted from embryos in a multiprotein complex of approximately 600 kDa. In contrast, the 18 and 27 kDa proteins predicted from the sequence of cloned cDNA to be transcribed and translated from the XIHbox 1 gene could not be detected, suggesting that they are rare or unstable in embryos. These data suggest that the new protein is involved in the development of Xenopus embryos, with a function possibly related to that of the homeoprotein XIHbox 1.
Fig. 2. Immunobloning analysis of Xenopus embryos extracts with
antibodies against XIHbox 1 protein. IAI Extracts from Xenopus embryos
were fractionared by electrophoresis on 18% polyacrylamide 50S
gels and analyzed by western blotting using different sera, pi, and PI/
preimmune sera from rabbits 1 and 2, i, and i]: immune sera from rabbits
1and 2: Brand B2: two different sera directed against E. co" B-galactosidase.
181 Three different extracts from Xenopus embryos were analyzed by
western blotting with serum from rabbit 2. Lane 1. extraction of proteins
with a buffer contaimng 50 mM KCI: lane 2. extraction wIth a buffer
con taming 250 mM KCf; lane 3. extraction by boifing embryos in 2% 50S,
0.1 M OTT. Composition of extraction buffers are given in Mateflals and
Methods.ICI Immunoblottmganalysls of Xenopus embryos extracts using
affinity-purified antibodies against B-gal-XIHbox I fusion protein. 50S
extracts (lane 1) and 50 mM KCf extracts (lane 2) from Xenopus embryos
were analyzed as in A and B, except that affinity-purified antibodies were
used in place of total rabbit serum.IDl/mmunoblottinganalysis of extracts
from Xenopus embryos on high resolution gels. The experiment ISidentical
to the previous ones with 50 mM KCf extracts. except that electrophoresIs
was on a high resoluMn 6% polyacrylamide 50S gel to better analyze the
doublet at 115 kOa. Lane 1. control with anti l3-galactosidase serum; fane
2, total rabbit serum: lane 3. affinity-purified antIbodies. Molecular weight
markers were LMWfrom Pharmacla. and E. coil B-galactosldase (116 kDa)
from Sigma.
Fig. 3. Analysis of Xenopus embryo extracts with antibodies specific to different domains of XIHbox 1 protein. Antibodies specific for 3 different
domains of the XIHbox 1 protein were purified from total rabbit serum using 3 different affinity columns prepared with fusion proteins containing the
amino-terminal region, the central homeodomain, or the carboxy-terminal region of the total protein. (AI Affinity-purified antibodies to one domain do
not bind to the other 2 domains. Three identical 50S-polyacrylamide gels were loaded with fusion proteins N, Hand C, and analyzed by western blotting
using affinity-purified antibodies anti-N, anti-H, and anti-C, as indicated. A similar gel containing in addition pure maltose binding protein (Mr 42,000) was
analyzed with total rabbit serum; In this case, bands in the upper part of the gel are due to l3-galactosidase. (BJ Control of the affinities of the purified
antibodies. Three western blots containing fusion protein T (with an extra band at -48 kDa due to premawre termination of translation in the
homeodomain, see Materials and Methods; this band only appears with anti-N) and equal amounts of fusion proteins N, H, and C were probed with affinitypurified
antibodies against domains N, H, and C, respectively. A fourth blot containing all 4 proteins was probed with total rabbit serum. On this latter
blot, as in panel A. the bands of high molecular weight are due to l3-galactosidase. tC) Analysis of Xenopus embryo extracts with the affinity-purified
antibodies. Six identical blots prepared with proteins extracted from Xenopus embryos at different stages of developmen t were probed with the following
antibodies: affinity purified anti-N, anti-H, anti-C, total immune serum, anti f3-galactosidase, and preimmune serum. Stages of development: 4,8-9, 10-12,20-21,22,23-31, in lanes 1-6, respectively.
Fig. 4. Immunolocalization of the 115 kDa
protein in Xenopus embryos. The 3 affinity-
purified antibodies anti-N, anti-H, and anti-
C were used in immunohistochemistry experiments
to localize the protein in who/emount
embryos at stage 25 of development
(A) or in serial sections of embryos. A transversal
section at the trunk level of a stage 25
embryo is shown IS), other sections gave
similar results.
Fig. 5. Variation of the band pattern in embryos originating from
different parents. Extracts from Xenopus embryos at stage 22 of development
but originating from 7 different parent couples (lanes 1-7) were
analyzed by immunoblotting using affinity-purified antibodies against total
XIHbox 1 protein.
Fig. 6. Variation of the band pattern during the course of development.
Embryos originating from 2 different couples (couples number 3 and
4 in Fig. 5) were collected at the indicated stages of development and
protein extracts were analyzed by Immunoblotting using affinity-purified
antibodies agamst total XIHbox1 protein.
Fig. 7. Analysis of extracts from Xenopus embryos by gel filtration. A
protein extract from embryos was fractionated on a Superose 6 column.
The top of the Figure shows the optical density of the fractions at 280 nm,
and a calibration curve of the column obtained with proteins of known
molecular weights. Fractions were analyzed by immunoblotting using total
immune serum, as shown by the western blots at the bottom of the Figure.
Lanes labeled C are controls loaded with the starting protein extract. The
115 kDa protein is present In fraction 16 (excluded volume) and predominantly
in fractions 25-26, the bands of lower molecular weights in fractions
29-34 do not stain with affinity-purified antibodies.
