Fig 3.

MCPyV ST interacts with NEMO. (A) 293 cells were cotransfected with either EGFP or EGFP-ST and the indicated GST-tagged eukaryotic expression vectors. After 24 h, cell lysates were incubated with GST-Sepharose 4B beads, and interacting proteins were immunoblotted with GFP-specific antibodies. (B) 293 cells were cotransfected with EGFP or EGFP-ST in the presence of either GST or GST-NEMO expression vectors. (i) Transfected cell lysates were probed with GFP-, FLAG-, and GST-specific antibodies to serve as a loading control (inputs). (ii) Transfected cell lysates were then incubated with either beads alone control, control antibody (Ab), or GFP-TRAP affinity beads, and bound protein was immunoblotted with a GST-specific antibody in addition to a GFP-specific antibody as a precipitation control. (C) (i) i293-ST cells remained uninduced or were incubated with doxycycline for 48 h. Cellular lysates were immunoblotted with a FLAG-specific antibody. (ii) Cellular lysates were then incubated with polyclonal NEMO-specific antibody bound to protein A-Sepharose beads, and precipitated proteins were then immunoblotted with a FLAG-specific antibody in addition to monoclonal NEMO-specific antibody as a precipitation control.