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Fig. 1. Characterization of different Shh proteins in rat E11 brain and limb
tissue. (A) Comparison of different N- and C-terminal lipid-containing Shh
proteins expressed by stably transfected cell lines and embryonic tissue. The
neural cell line C17 was transfected with different Shh constructs in order to
obtain stable cell lines expressing different N- and C-terminal lipids.
Whole-cell lysates from stably transfected cells were separated by SDS-PAGE
and used to compare the migration of Shh proteins present in E11 embryonic
tissues. Western blot analysis using anti-Shh (1-10, 11-16) or anti-tubulin
(7'-10' antibodies). (Lane 1) uShhN (7 ng; Shh made in E.
coli containing no N- or C-terminal lipids). Lanes 2-5 show lysates of
C17 cells stably expressing different lipid-containing Shh proteins (described
in Table 1). (Lane 2) wtShh
(N+C lipid); (lane 3) C24S-Shh (C lipid alone); (lane 4,) C24S-ShhN (no lipid,
10-fold more cell lysate was loaded in order to detect cell-associated
protein); (lane 5) ShhN (N lipid alone). (Lane 6) uShhN (5 ng). (Lane 7) Rat
E11 midbrain/hindbrain (MB/HB) tissue lysate. (Lane 8) Rat E11 MB/HB lysate
diluted 4-fold in denaturing lysis buffer containing 1% SDS. (Lane 9) Rat E11
forebrain (FB) tissue lysate. (Lane 10) Rat E11 limb tissue lysate. (Lanes
7'-10') Lanes 7-10 were stripped and re-probed with anti-tubulin
antibody as a loading control. (Lane 11) uShhN (10 ng). (Lane 12) E11 rat
MB/HB tissue lysate. (Lane 13) E11 rat FB tissue lysate. (Lane 14) Lysate of
C17 cells stably expressing C24S-Shh protein. (Lane 15) Rat E11 embryo tissue
lysate with brain, limbs and branchial arches removed. (Lane 16) Rat E11 limb
tissue lysate. Lanes 11-16 were exposed for a shorter amount of time. Size
standards are shown on the left. The three arrows on the right highlight the
migration of different Shh proteins in the range of those detected in C17
transfected lysates. The asterisk marks a form of Shh that is present in the
brain and absent from limb tissue. (B) A comparison of the profile of Triton
X-100 extractable membrane Shh proteins containing different N- and C-lipids
from Shh stably transfected cells, and from embryonic midbrain/hindbrain and
limb tissues. Triton X-100 extracted membranes were analyzed by western
analysis using anti-Shh antibody. Membranes isolated from Shh stably
transfected cell lines (lanes 1-7), E11 rat embryonic midbrain/hindbrain
(lanes 8-14) or E11 rat embryonic limb (lanes 16-22) were treated with
increasing amounts of Triton X-100: 0.1% (lanes 1,8,16), 0.2% (lanes 2,9,17),
0.4% (lanes 3,10,18), 0.6% (lanes 4,11,19), 0.8% (lanes 5,12,20), 1% (lanes
6,13,21). (Lanes 7,14,22) Pellet; (lanes 15,23) uShhN, 10 ng.
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