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. 2011 May 13;286(26):23608–23619. doi: 10.1074/jbc.M110.206474

FIGURE 5.

FIGURE 5.

Variable biological activities of ShhNp CW mutants. A, C3H10T1/2 osteoblast precursor cells were incubated with wild-type and CW mutant ShhNp conditioned media, and the relative amount of Shh-induced AP activity was determined as a readout for biological activity. Medium obtained from mock-transfected Bosc23 cells was used as a control (mock). ShhNp-induced AP activity was entirely blocked by the teratogen cyclopamine (CA) (2.5 μm, p ≤ 0.001, n = 2), a specific inhibitor of Shh signaling. Consistent with the previously observed loss of biological activity of monomeric CW-deficient morphogens, ShhNp5xA and ShhNpΔCW were also inactive in this assay. In contrast, both ShhNpK33E/R34L and ShhNpR34A/K38A retained significant biological activities in this assay if compared with the mock control (p < 0.001, n = 3), consistent with preserved HS binding of the mutant multimeric morphogens. *, p = 0.044, n = 3. n.s., not significant (p = 0.13, n = 3). One representative result of three independent experiments is shown. B, mutant and wild-type morphogens were subjected to immunoprecipitation using the conformation-dependent antibody 5E1 coupled to protein A-Sepharose, and the immune precipitates were subjected to SDS-PAGE and immunoblotting. For normalization of different expression levels, heparin-Sepharose pull-downs of the same expressions served as controls. 5E1 reactivity was detected toward all morphogens, demonstrating that loss of ShhNp5xA and ShhNpΔCW biological activities was not due to misfolding of the mutated morphogens. C, dose-dependent induction of C3H10T1/2 osteoblast precursor cell differentiation. C3H10T1/2 cells were incubated with equivalent amounts of wild-type or CW mutant ShhNp, and AP activity was measured at different morphogen concentrations as a readout for Shh-induced C3H10T1/2 differentiation. ShhNp5xA and ShhNpΔCW remained inactive at all concentrations used, and ShhNp, ShhNpK33E/R34L, and ShhNpR34A/K38A induced C3H10T1/2 cell differentiation in a dose-dependent manner. Cyclopamine was added as a control (2.5 μm).