Fig. 2: The ASCPYD-specific protein binder effectively disassembles preformed ASC specks.
A The ASC levels in each fraction were obtained through sucrose-gradient (5–45%) ultracentrifugation of recombinant ASC specks. ASC specks were prepared by TEV cleavage of 5 μM MBP-fused full-length ASC, followed by the addition of rB7 as designated molar ratio for 1 h and ultracentrifugation (42,000×g, 10 min). rOff indicates off-target protein binder (wild-type repebody). B Negative stain EM images of recombinant ASC specks after treatment with rB7 or rOff. ASC specks are prepared by the same method as in (A). Low (15 μg/ml) and high (50 μg/ml) concentrations of rB7 or rOff were added to 30 μg/ml of preformed ASC specks for 30 min. Bars indicate 100 nm. C Changes in the levels of mNeonGreen-fused ASC specks in the supernatants (SN) and pellet (PE) after centrifugation (18,000×g, 20 min) over time. mNeonGreen-fused ASC specks were expressed and purified from HEK293 cells. ASC speck particles were diluted to ~5 × 105 per μl in DPBS, followed by incubation with 10 μM of rB7 or rOff, and the relative amounts of ASC in the supernatants and pellet were analyzed through a western blot. Treatment of ASC specks with DPBS was used as the negative control. Data were representative of three independent experiments. D. Western blot analysis of cross-linked mNeonGreen-fused ASC specks after incubation with 10 μM of rB7 or rOff. Samples obtained at designated time intervals were cross-linked by 2 mM DSS for 30 min. Data were representative of three independent experiments.
