Fig. 5.

14-3-3 family members bind to DCBLD2 in an ABL-induced manner.A, DCBLD2-FLAG-MYC was transiently expressed in 293 cells in the presence and absence of ABL. GST-fusions with 14-3-3σ, 14-3-3ε, and 14-3-3ζ on GSH resin were incubated with 293 extracts in pulldown assays. Bound proteins were denatured and analyzed by SDS-PAGE and Western blotting. Ponceau stains display levels of GST-14-3-3(X) and α-MYC blots of the pulldowns conditions in which DCBLD2 bind to the fusion protein. Immunoprecipitations (IP, α-FLAG) show DCBLD2 expression levels. Whole cell extracts (WCEs) show levels of ABL expression (α-ABL) and the tubulin blot serves as a loading control. B, DCBLD2-FLAG-MYC was transiently expressed in 293 cells in the presence and absence of ABL. Immunoprecipitations (α-MYC) from 293 cell lysates were subject to SDS-PAGE and far-Western blotting with eluted GST-14-3-3 fusions proteins, followed by α-GST primary antibodies. Signals in α-GST blots show relative levels of GST-fusions interacting with DCBLD2 in the presence and absence of ABL, and the α-MYC blot displays levels of DCBLD2 protein. C, α-GST signals from (B) were quantified across three replicates and normalized to DCBLD2 levels using densitometry. Fold changes of GST-14-3-3 binding in the presence of ABL are plotted. Error bars are representative of three trials.