Figure 5.

Characterization of Interaction between ChREBP and 14-3-3

A and B, Poly-His pull-down assay. The 832/13 cells stably expressing 6×His/c-myc-tagged 14-3-3β and c-myc-tagged ChREBP or its R128 mutant were treated with low (L, 2.5 mm) or high (H, 27.5 mm) glucose for 4 h and then either directly lysed for the pull-down assay (A) or treated with DSP before lysis for pull-down assay (B). C, Poly-His pull-down assay. pCHM-14-3-3β was cotransfected with plasmids expressing indicated wild-type or mutant ChREBP into 832/13 cells. Cells were lysed after 24 h for poly-His pull-down assay. D, GST pull-down assay. Glutathione-Sepharose beads coated with the indicated recombinant protein were incubated with recombinant c-myc-tagged 14-3-3β. The amount of 14-3-3β retained on these beads was examined by Western blot with anti-c-myc antibody. WT, Wild type.