FIGURE 6.

RET interacts with ATF4. A, in vitro GST pull-down assay with ³⁵S-labeled RET-C634W proteins in the presence of GST and GST-ATF4. B–D, interactions between RET and ATF4 in cells; B, FRET between RET and ATF4 was measured using FLIM after transfection in HEK293T cells treated with 5 μm MG132 for 16 h. Confocal images and co-localization of the same field are shown (left). The histograms as shown (right) indicate the average fluorescence lifetime distributions corresponding to cells in the field of view. Lifetime images were generated by pixel-by-pixel mapping of the lifetime data and are represented as false color images adjacent to histograms. Scale bars are indicated by the white line. C, ATF4 was co-purified with RET. HEK293T cells were transfected with His-RET- kinase domain (residues 657–1114) and FLAG-ATF4 followed by purification of RET with nickel beads and then Western blot (WB) with ATF4 or His tag antibodies. D, RET was co-immunoprecipitated (IP) with ATF4. FLAG-ATF4 and HA-tagged full-length WT RET were expressed in HEK293T cells followed by reversed immunoprecipitation and Western blot with the indicated antibodies.