Figure 6.

PDR-1 loss-of-function fails to ubiquitylate CED-10. (a) HEK293T cells were transfected with the indicated expression plasmids, and 500 μg of lysates were used to purify overexpressed FLAG-PDR1 using a FLAG-agarose resin. Immune complexes were immunoblotted for CED-10 and PDR-1 using anti-GST and anti-FLAG antibodies, respectively. Similar results were obtained in three independent experiments. (b) One microgram of HEK293T cells overexpressing FLAG-PDR1 was immunoprecipitated with anti-FLAG-agarose beads. Immune complexes were immunoblotted for human endogenous Rac1 and PDR-1 using anti-Rac1 and anti-FLAG antibodies. (c and d) Cells overexpressing the indicated plasmids were treated with 20 μM MG-132 for 18 h. His-tagged ubiquitin was purified using using Ni2⁺-NTA-agarose beads and the ubiquitylated forms of CED-10 were visualized by immunoblotting using an anti-GST antibody