Identification of ASC isoforms. (A) Differentiated THP-1 macrophages were separated into nuclear and cytosolic fractions and analyzed for ASC expression using a monoclonal anti-ASC antibody recognizing the PYD of ASC by immunoblot. Blots were stripped and re-probed with antibodies for the cytosolic GAPDH and nuclear Lamin A to control for fractionation efficiency. (B) THP-1 lysates were analyzed by immunoblot for ASC expression using antibodies recognizing the PYD, the linker, and the CARD, respectively. (C) Lysates from PMA-differentiated and LPS-treated (300 ng/ml) THP-1 cells and J774A1 cells were separated by SDS/PAGE and immunoblotted with a PYD-specific anti-ASC antibody (AL177). (D) Lysates of human peripheral blood macrophages (PBM) that were left untreated, or treated with LPS for the indicated times, were immunoblotted for ASC. (E) PMA-differentiated THP-1 cells were treated with LPS (300 ng/ml) for the indicated times and analyzed by RT-PCR for ASC transcripts using the primer pairs pr-1 (ASC, 299 bp; ASC-b, 242 bp), pr-2 (ASC-c, 66 bp), and pr-3 (ASC and ASC-b, 128 bp; ASC-d, 100 bp). A short exposure (upper panel) and long exposure (middle panel) is shown, because of the relative low abundance of ASC-d transcripts. A β -actin primer pair (533 bp, lower panel) was used as a control.