Figure 6. cNICD half-life in the PSM is increased after exposure to Roscovitine, DRB and PHA-767491.

(A): Endogenous cNICD levels analysed by Western Blot (after normalisation to alpha tubulin loading control) in PSM explants cultured initially in the presence of each reagent for 3 hr and subsequently in the presence of inhibitor plus cycloheximide for an additional 30 min before the time-course. Panels show log transformation on repeated data. The half life is proportional to the inverse of the inferred slope (Equation [4]). (B) Bar chart showing the half-life of cNICD in each condition. Asterix represents statistically significant differences p < 0.05. (C) Left Panel: Treatment in the presence (+) or absence (−) of MLN4924 for 3 hr reveals that the inhibitor treated explant has lagging expression of cLfng compared to the control contralateral explant. Right panel: After 3 hr in the presence of MLN4924 one PSM explant was fixed while the other was treated for another 45 min, showing cLfng expression is still dynamic in the presence of this inhibitor. (D) Following the assay described in Figure 4, levels of cNICD, normalised to levels of alpha tubulin, in the chick PSM pools are highly increased in the presence of MLN4924. Western Blot analysis of pooled PSM lysates from half PSM explants treated ‘+’ or ‘−’ MLN4924 reveals levels of phosphorylated β-catenin are increased in the chick PSM after 3 hr treatment with MLN4924.

DOI: http://dx.doi.org/10.7554/eLife.05842.013

Figure 6—figure supplement 1. Roscovitine, PHA-767491 and DRB treatment increases the level of cNICD in the chick PSM.

Figure 6—figure supplement 1(A–B): Western Blot analysis to monitor the decay of cNICD protein: after 3 hr treatment with the reagents the pools of 9 explants were exposed to cycloheximide for 30 min to inhibit new protein synthesis, and subsequently the decay of cNICD protein was monitored whereby one pool was removed from culture and protein lysate prepared while the corresponding pool was cultured for an additional 30–60 min before lysate also prepared. The +/− lanes with Cycloheximide (CHX) show that levels of cNICD fall dramatically in the absence of de novo protein synthesis in the presence of this reagent.

DOI: http://dx.doi.org/10.7554/eLife.05842.014