PDK1 is mono-ubiquitinated in a variety of cell lines.

<p><b>A</b>: HEK293T cells were transfected with 6×His-tagged ubiquitin and ubiquitinated proteins were isolated with nickel beads under denaturing conditions. Pull-downs and input were analyzed with an antibody recognizing PDK1. Ub-PDK1 indicates ubiquitin-modified PDK1 species; asterisk indicates unspecific cross-reacting band. <b>B</b>: HEK293T cells were transfected with a V5-tagged PDK1 cDNA and V5-PDK1 was immunoprecipitated. Immunoprecipitations (IP) and input were immunoblotted with a V5 antibody. <b>C</b>: HEK293T cells were co-transfected as indicated with HA-tagged ubiquitin and V5-PDK1. Immunoprecipitation of HA-ubiquitin and input were immunoblotted with a V5 antibody. <b>D</b>: HEK293T cells were co-transfected as indicated and whole cell extracts were probed with anti-V5 or anti-HA antibody. <b>E</b>: HEK293T cells were transfected with V5-tagged PDK1 or a linear PDK1-ubiquitin C-terminal fusion protein. The whole cell extracts were probed with anti-V5 antibody. <b>F</b>: Endogenous PDK1 was immunoprecipitated from HEK293T cells and blotted with anti-PDK1 antibody. The following controls were used: anti-PDK1 antibody only, sepharose beads only, anti-lgG2a control antibody with beads. <b>G</b>: Lysates from the indicated cell lines were subjected to PDK1 immunoprecipitation. Immunoprecipitations were immunoblotted with a PDK1 antibody.