FIGURE 5.

Multiple lysine residues are required for DDIT4 regulation. A, schematic demonstrating positions of lysine residues on DDIT4. B, 24 h after transfection HeLa cells were treated with either MG132 or DMSO for 6 h. WT and variant DDIT4-HA levels were assessed by immunoblot analysis. C, HeLa cells stably expressing two unique HUWE1 shRNAs were treated in the absence and presence of Dox for 48 h, transfected with WT and variant DDIT4-HA constructs for 24 h, and incubated with MG132 or DMSO for an additional 6 h. DDIT4-HA levels were determined by immunoblotting. D, DDIT4-HA constructs containing a single lysine residue were assayed as described in B. E, HeLa cells stably expressing inducible HUWE1 shRNA 1 were transfected with DDIT4-HA constructs containing a single lysine residue and analyzed as described in C. F and G, K129R/K155R/K188R and K218R/K219R/K220R DDIT4 constructs were analyzed as in B and C, respectively.