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. 2011 May 3;21(9):1343–1357. doi: 10.1038/cr.2011.76

Figure 3.

Figure 3

Temperature-sensitive and calcium-dependent degradation of IhhN proteins. (A) Stability of WT or mutant IhhN proteins in EcR-CHO cells cultured for various durations at 30 °C and 37 °C. D100E mutant IhhN protein is degraded more rapidly than the others at 37 °C. All proteins are stable at 30 °C. (B) D100E IhhN is detectable in the conditioned medium from transiently transfected EcR-CHO cells at 37 °C, when the cholesterol modification is disrupted. The primary antibody indicated above is an anti-FLAG monoclonal antibody. (C) Wild type, E95K, and D100E mutant protein expressions were analyzed in the presence of increasing calcium concentrations (8-64 mM). Their relative stabilities were significantly enhanced. (D) Wild type IHH protein expression was assayed in the presence of increasing EGTA concentrations (0.1-0.8 mM). The level of IhhN protein was markedly reduced in the presence of 0.4 mM EGTA and was barely detectable in the presence of 0.8 mM EGTA.