Xenbase Image ID: 125818

Figure 2. Hh processing in Xenopus egg extracts. (A) The in vitro translated 35S-labeled Xenopus Sonic Hh (XShh) wild-type (WT) precursor was incubated at room temperature with Xenopus egg extracts for the indicated times. Parallel experiments were performed with three point mutants. The samples were analyzed by SDS-PAGE and autoradiography. The graph shows the quantification of the Hh precursor. (B) As in A, but the extract was supplemented with the indicated concentrations of oxidized or reduced glutathione (GSSG or GSH). (A and B) n = 3 time points. (C) In vitro translated 35S-labeled XShh fused at its N terminus to the maltose-binding protein (MBP-XShh) was incubated at room temperature with Xenopus egg extracts for the indicated times, in the absence or presence of 0.5% of either the cholesterol-sequestering detergents digitonin or cholate, or the control detergent Triton X-100 (TX-100). The samples were analyzed by SDS-PAGE and autoradiography. (D) In vitro translation was used to generate 35S-labeled fusions of MBP and either the N-terminal fragment of XShh (N), the C-terminal fragment of XShh (C), full-length XShh (FL), full-length XShh with a cysteine mutation in the active site (C199A), or XShh lacking the last 93 amino acids (ΔC). The fusions were incubated for 1 h with Xenopus egg extracts and subjected to Triton X-114 partitioning. Aliquots of the input (T), of the aqueous phase (A), or of the detergent phase (D) were analyzed by SDS-PAGE and autoradiography. (E) As in A, but with an MBP fusion of either wild-type XShh (FL) or a mutant lacking the last 93 amino acids (ΔC). Molecular masses are given in kilodaltons. Image published in: Chen X et al. (2011) © 2011 Chen et al. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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