Figure 2. H486R optineurin is defective in interaction with CYLD.

Yeast strain PJ69-4A was co-transformed with wild-type optineurin or its mutants and CYLD. Transformants were grown on selection media lacking adenine to assay interaction. GST-optineurin, GST-H486R or GST alone bound to glutathione agarose beads were incubated with lysates of HeLa cells transfected with HA-CYLD. The bound proteins were eluted and immunoblotted with anti-HA antibodies. Western blot was done with optineurin antibody (lower panel) to confirm the identity of the GST fusion proteins. HeLa cells were co-transfected with HA-CYLD and Myc-OPTN or Myc-H486R. Lysates were immunoprecipitated with anti-HA antibody and subjected to western blotting. WCL, whole cell lysate.