Figure 7. COPs selectively interact with caspase-1 and ASC by a competitive binding mechanism.

(A and B) GFP, GFP-tagged CARD16, CARD17, and CARD18 expressing, and (A) CASP1^KO or (B) ASC^KD THP-1 cells were left untreated, primed with Pam3CSK4 (Pam) (1 μg mL⁻¹, 3 h), activated with nigericin (Nig) (2.5 μM, 5 min) as indicated, and were subjected to immunoprecipitation (IP) with immobilized (A) anti-caspase-1 or (B) anti-ASC antibodies and analyzed alongside total cell lysates (TCL) by SDS-PAGE and immunoblot.

(C) HEK293 cells were transfected with caspase-1^{CARD-mNeonGreen}, caspase-1^{CARD-mScarlet-I}, CARD16, CARD17, and CARD18 as indicated, and emission scans were collected from 490 to 750 nm in 2-nm increments using a 470-nm excitation wavelength and presented as fluorescence intensity (n = 2).

(D) PMA-differentiated control (Ctrl), CARD16, CARD17, CARD18 expressing, and CASP1^KO and ASC^KO THP-1 cells were left untreated, primed with Pam (1 μg mL⁻¹, 3 h), activated with nigericin (Nig) (10 μM, 15 min) as indicated, and subjected to proximity ligation assay (PLA) between caspase-1 and ASC and analyzed by fluorescence microscopy. Results are presented as fold PLA⁺ cells compared with primed Ctrl cells and normalized to DAPI⁺ cells (n = 9–15, mean ± SD). *p < 0.05.

See also Figure S5.