MEKK1-Dependent Apoptosis Requires the PHD Figure 3. Creation and characterization of MEKK1 mutations. A. Schematic diagram of MEKK1, and MEKK1 mutants illustrating the plant homeodomain (MutP), the ubiquitin-interacting motif (delU), the kinase domain (MutK) and the putative caspase cleavage site (MutC). Arrows indicate the mutations created within each domain. B. Proper protein expression and determination of downstream signaling via c-Jun phosphorylation after overexpression of each mutation. Each mutant was transiently transfected into 293T cells in the highly expressing pApuro construct. Cell lysates were run on SDS-PAGE, western blots were performed and MEKK1 was detected with aMEKK1 C-22. Blots were next probed with total c-Jun, which recognizes both the dephosphorylated and phosphorylated forms of c-Jun. C. Cellular localization of each mutant. HeLa cells were transfected with pApuro MEKK1, MutP, delU, MutK or MutC and stained with DAPI (nucleus) and aMEKK1 C-22 antibody (MEKK1) after 24 hours of transfection. D. The PHD mutant does not colocalize with lysosomes to a greater extent than wild type MEKK1. HeLa cells were transfected with flag-MEKK1 or flag-MutP and stained with DAPI, M2 aflag (MEKK1) and LAMP1 (lysosomes) after 24 hours. MEKK1 was detected with mouse aFITC and LAMP1 with rabbit aTRITC. E. The PHD mutant localization is not altered by vinblastine treatment. HeLa cells were transfected with pApuro MEKK1, MutP and MutK. After 24 hours cells were treated with vinblastine for one hour. MEKK1 was visualized with aMEKK1 C-22 antibody and nuclei visualized with DAPI. doi:10.1371/journal.pone.0017310.g003 downstream signaling, as assessed by phosphorylated c-Jun, with the anticipated exception of the kinase mutation (Figure 3B). To determine the cellular localization of each mutant, we expressed each in HeLa cells and visualized localization with indirect immunofluorescence (Figure 3C). The localization of MEKK1 has been well characterized by immunofluorescence. As previously published and in our system, MEKK1 was expressed diffusely through the cytosol and in small punctuate structures [21]. The UIM, caspase 3 and kinase dead mutants were expressed in a manner similar to wild type MEKK1, but the PHD mutant was located expressly in large, distinct punctuate structures. To ensure that the change in the PHD mutant localization was not due to enhanced lysosomal degradation, indirect immunofluorescence was performed with both MEKK1 and LAMP1, a marker for lysosomes (Figure 3D). We found that neither wild type MEKK1 nor the PHD mutant significantly colocalize with lysosomes. To investigate whether the subcellular localization of MEKK1 or any of the MEKK1 mutants change upon treatment with MTIs, transfected HeLa cells were treated with vinblastine and subjected to immunofluorescence as before PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e17310