Fig. 1S. Dab2 overexpression stabilizes Axin expression.
(A) F9 cells were transfected with a Myc-tagged Axin expression construct in the +/- of Flag-tagged Dab2.
Following transfection, cells were pulse labeled with [35S]-methionine for 3h and chased with cold media for
the times indicated. Cell lysates, adjusted for equal precipitable counts, were subjected to
immunoprecipitation with -Myc monoclonal antibody, separated by SDS-PAGE, and analyzed by
autoradiography. Half-life of Myc-tagged Axin is ~4h and disappears at ~8h. In the presence of ectopically
expressed Dab2 however, Axin expression is stabilized. The half-life of the protein is greater than 8h and
the mobility shift of the labeled band is not as apparent. (B) Levels of Dab2 protein as in (A) from 3
separate experiments were determined by densitometric scanning and quantitated using a STORM
scanner and ImageQuant software from Molecular Dynamics. Levels are expressed as a percentage of
protein remaining at the various time points, with levels at time 0 designated as 100%. The data are
presented as means +/- standard deviation. (open circle; -Flag-tagged Dab2) and (closed circles; +Flag-
tagged Dab2). (C) F9 cells were co-transfected with Myc-tagged Axin and increasing concentrations of
Flag-tagged Dab2. Following transfection, cells were pulse labeled with [35S]-methionine for 3h and cell
lysates, adjusted for equal precipitable counts, were subjected to immunoprecipitation with -Myc
monoclonal antibody, separated by SDS-PAGE, and analyzed by autoradiography. The data demonstrate
that increasing concentrations of co-transfected Dab2 result in increased Myc-Axin that is labeled over the
3h [35S]-methionine labeling period.
F9 cells in 10 cm plates were transfected with Myc-tagged Axin as described above. After 24h, cells were
incubated in methionine and cysteine-free media for 30 min followed by metabolic labeling of the cells in
200 Ci/ml [35S]-methionine for 3h and either lysed immediately or chased in regular media for various
times. Transfected Myc-tagged Axin was immunoprecipitated with an anti-Myc antibody and analyzed by
SDS-PAGE and autoradiography.