Figure 3.

The 20S proteasome generates CARD8^p44in vitro. A, purified CARD8^FL WT protein were incubated at varying dilutions with or without purified 20S proteasomes for 4 h. Reactions were quenched with 2× loading dye prior to immunoblotting analysis. B, purified 20S proteasomes were preincubated with the indicated vehicle or proteasome inhibitors for 1 h, prior to adding purified CARD8^FL WT for an additional 1 h. Reactions were quenched with 2× loading dye prior to immunoblotting analysis. C, purified CARD8^FL and CARD8^ZUC (each 800 nM) were incubated with purified 20S proteasomes (100 nM). At the indicated timepoints, aliquots were removed from the mixture, quenched with 2× loading dye, and analyzed by immunoblotting. D–F, the indicated purified proteins were treated and analyzed as described in (A). Immunoblots are representative of three or more independent experiments.