Cleavage of FMF-associated mutant pyrins by caspase-1 and cellular localization of cleaved fragments. (A) Myc-tagged D330A, WT, or FMF-associated mutant pyrin was transfected into PT67 cells. After 24 hours, cell lysates (10 μg total protein) underwent Western blotting with antipyrin Ab (top). The same amounts of lysates were incubated with 20 units of recombinant caspase-1 for 10 minutes at room temperature. The incubated cell lysates were subjected to Western blotting with antimyc, detecting the C-terminal cleavage fragment (middle), and antipyrin Abs, detecting the smaller N-terminal cleavage fragment (bottom). (B) GST-fused WT or mutant pyrin was cotransfected into PT67 cells with (lanes 1-5) or without (lanes 6-9) caspase-1. Cell lysates were analyzed by Western blots using antipyrin Ab (top panel). The cleaved and uncleaved pyrin fragments were quantified by densitometry and represented as percentage of total pyrin (bottom panel). (C,D) Lysates of PBMCs from 10 healthy individuals (C) and 10 FMF patients (D) were subjected to SDS-PAGE, and Western blotting with antipyrin Ab. Exposure times were the same for panels C and D. The full-length and cleaved pyrin were quantified by densitometry and represented as percentage of total pyrin. (E) HeLa cells were transfected with myc-tagged full-length pyrin (i-iii), N-terminal cleaved pyrin (iv-vi), or C-terminal cleaved pyrin (vii-ix). After 24 hours, cells were fixed with 4% paraformaldehyde in PBS, and stained with AlexaFluor 488–conjugated antimyc Ab (green), counterstained with DAPI (blue), and visualized on a Leica DMR microscope. Pyrin variants are shown in the first column (i, iv, and vii); nuclei are in the second column (ii, v, and viii); merged images are in the third column (iii, vi, and ix).