Fig 8.

MCPyV ST colocalizes with NEMO and cellular phosphatase subunits PP4C and PP2A Aβ in discrete cytoplasmic puncta. (A) Huh7 cells were transfected with either EGFP or EGFP-ST in the presence of Myc-NEMO and FLAG-PP4C expression vectors. After 24 h, cells were fixed and permeabilized, and GFP fluorescence was analyzed by direct visualization, whereas NEMO and PP4C were identified by indirect immunofluorescence using Myc- and FLAG-specific antibodies, respectively. (B) A similar experiment was repeated, but cells were transfected with either EGFP or EGFP-ST in the presence of EE-PP2A Aβ expression vectors. GFP fluorescence was analyzed by direct visualization, whereas endogenous NEMO and PP2A Aβ were identified by indirect immunofluorescence using NEMO- and EE-specific antibodies, respectively. (C) 293 cells were cotransfected with EGFP (i) or EGFP-ST (ii) in the presence of NEMO-myc and either control or FLAG-PP4C or EE-PP2A Aβ expression vectors. (i) Transfected cell lysates were probed with GFP-, Myc-, FLAG-, or EE-specific antibodies to serve as a loading control (Inputs). Transfected cell lysates were then incubated with Myc affinity gel, and bound protein was immunoblotted with a GFP-, FLAG-, or EE-specific antibody in addition to a NEMO-specific antibody as a precipitation control.