Figure 2.

Selenite treatment triggers deubiquitination of K63 polyubiquitin chains on RIP1 and subsequent DISC formation. (a) RIP1 coimmunoprecipitated with caspase-8 and FADD after selenite treatment. The results from the same cell lines were acquired by incubating different antibodies to different parts of the same membrane in the same experiment. An antibody against RIP1 was applied after stripping the polyubiquitin antibody to confirm equal loading. (b) Control or selenite-treated cells were stained with anti-RIP1 (CY3) and anti-caspase-8 (FITC) antibodies and subjected to confocal microscopy. The nuclei were stained with DAPI. The line furthest to the right is a magnification of the boxed area in the merged panel. Scale bar, 20 μm. (c) K63 ubiquitin-conjugated RIP1 was isolated by IP using an anti-RIP1 antibody followed by immunoblotting (IB) detection using anti-K63-polyubiquitin (right panel). The levels of total ubiquitinated cellular proteins were determined through direct IB in the same experiment (left panel). RIP1 was probed by IB to confirm equal loading. The results from the upper and lower panel were derived from the same experiment, with different exposure times. All experiments were performed in triplicate, and representative imagines are shown