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.