Molecular Cloning of ASC Isoforms

A search of the publicly available expressed sequence tag (EST) database revealed three potential ASC isoforms: ASC-b (Acc. No. BM456838), ASC-c (Acc. No. BE560228), and ASC-d (Acc. No. BM920038). The complete open reading frame of each isoform was subsequently amplified by PCR from pooled THP-1 cell cDNAs that were induced with a cocktail of cytokines for 2 to 24 hours. ASC-b, ASC-c, and ASC-d were amplified using the common forward primer 5'-CGGAATTCGATCCTGGAGCCATGGG-3' and the common reverse primer 5'-CGCTCGAGTGACCGGAGTGTTGCTG-3' and cloned into a modified pcDNA3 vector (Invitrogen) in frame with an NH₂-terminal myc epitope tag. The CARD of caspase 1 was amplified by high fidelity PCR and cloned into pGex4T-1 (Novagen). All other expression constructs (ASC, pro-IL-1β, pro-caspase 1, NLRP3^R260W) have been previously described [37 (link)-39 (link)].