GFP-SseK3 preferentially interacts with non-ubiquitinated TRIM32.

<p>Sub-confluent A431 cells were transiently transfected with plasmids encoding GFP-SseK1, GFP-SseK2, GFP-SseK3 or Myc-TRIM32 alone, or both GFP-SseK and Myc-TRIM32. 16–18 h post transfection, cells were washed with chilled PBS and lysed on ice using TK lysis buffer. Equal amount of cell lysates were used for the immunoprecipitation using GFP-nanotrap beads. Immunoprecipitated complexes <b>(A)</b> and cell lysates <b>(B)</b> were resuspended in SDS sample loading buffer and boiled for 5 min before resolved by SDS-PAGE, and transferred onto Immobilon-FL membrane. After blocking in Odyssey blocking buffer, membranes were incubated with primary antibodies against GFP and Myc, followed by IRDye conjugated secondary antibodies. Fluorescence was detected by Li-COR Odyssey infrared imaging system. Untransfected cells were used as a negative control. Representative images from three independent experiments are shown.