Reduced caspase-1 generation and IL-1β processing in Pml^−/− macrophages. (A) PML deficiency did not affect the expression of NLRP3, ASC, and procaspase-1. The expression of PML, NLRP3, ASC, procaspase-1, and GAPDH proteins was determined in BMDMs from Pml^+/+ and Pml^−/− mice. Numbers to the left indicate molecular weight in kDa. (B-D) PML deficiency decreased active caspase-1 generation and IL-1β processing. Pml^+/+ and Pml^−/− macrophages were primed with LPS for 4 hours, followed by stimulation with ATP for 40 minutes (B), alum crystal for 4 hours (C), or MSU for 4 hours (D). Culture supernatants and BMDMs were collected before and after LPS activation, or after stimulation with ATP, alum crystal, or MSU. Total cell lysates (TCL) were generated from BMDMs at each stage. Proteins in supernatants (SUP) were precipitated by cold (–20°C) acetone. Both supernatant precipitates and TCL were resolved by SDS-PAGE. The contents of mature IL-1β (p17) and active caspase-1 (p10) in supernatants, and PML, NLRP3, procaspase-1, pro-IL-1β, ASC, and GAPDH in total cell lysates, were assessed by Western blotting. (E) Reduced cathepsin B processing in PML-null macrophages. Pml^+/+ and Pml^−/− macrophages were primed with LPS, followed by MSU stimulation as in (B). The levels of cathepsin B and active caspase-1 p10 in supernatants, and the contents of procaspase-1 and pro-cathepsin B in total cell lysates were determined. (A-E) Results shown were representative of at least 3 independent experiments.