Figure 1.

Axin-ΔC6 has a shorter half-life than full-length Axin. A) In Axin^ΔC6/+ E13.5 embryos (lanes 2 and 3) or MEFs derived from such embryos (lane 4), Axin-ΔC6 (*) is present at a much lower level than endogenous Axin (**) when both are detected with anti-Axin antibody. Lane 1 shows analysis of a wild-type (WT) embryo. Axin-ΔC6 is larger than WT, endogenous Axin because it contains 6 Myc tags. B) Myc-Axin-ΔC6 (in Axin^ΔC6/+ MEFs) turns over more rapidly than WT Myc-Axin (in Axin^Ax/+ MEFs). Axin^Ax/+ and Axin^ΔC6/+ MEFs were cultured with 20 μg/ml of cycloheximide (CHX) for the indicated number of hours. The level of WT or mutant Myc-Axin encoded by the modified Axin locus was measured by Western blotting using anti-Myc antibody at different time points. The band in the Axin-ΔC6, 9 h lane, above the position of Axin, is a background band (*). Graph shows the results of three independent experiments. C) Proteasome-specific inhibitor ALLN stabilizes Axin-ΔC6 more than WT Axin. Axin^Ax/+ and Axin^ΔC6/+ MEFs were incubated with 50 μM of ALLN for the indicated times, and anti-Myc Western blotting was performed. The results show that the level Axin-ΔC6 protein increases more than WT Axin when proteasome degradation is inhibited. Graph shows the results of two independent experiments. Graphs plot independent experiments in different colors: solid lines, WT Axin; dotted lines, Axin-ΔC6. The levels of Axin or Axin-ΔC6 protein at each time point were normalized to the starting level (100%).