Fig 6. Ectopic expression of SseK3 does not alter the ubiquitination of TRIM32.

Sub-confluent A431 cells were transiently co-transfected with plasmids encoding Myc-TRIM32 and GFP-SseK3 or GFP-SseK3AAA. Untransfected cells were included as a negative control. 16–18 hrs post transfections, cells were then washed and harvested in TK lysis buffer. Equal amounts of pre-cleared cell lysates were used for immunoprecipitation using mouse monoclonal anti-Myc antibody coupling with Protein G agarose beads. Immunoprecipitated proteins (A) and whole cell lysates (B) were boiled for 5 min in SDS sample loading buffer and resolved by SDS-PAGE/ western blots. Membranes were incubated with anti-ubiquitin or anti-Myc antibodies, whereas anti-tubulin antibody was included as a loading control. After the incubation with IRDye conjugated fluorescence secondary antibodies, fluorescence intensities were detected and scanned by using Li-COR Odyssey infrared imaging system. Represented images from three independent experiments were shown. The asterix (*) indicates 80 kDa of mono-ubiquitinated TRIM32.