Expression plasmids encoding NLRP3, NLRP3 cysteine mutants, and chimeric NLRP3 mutants, myc-ASC, GFP-ASC, caspase-1 (CASP1), and pro-IL-1β (IL1B) have been described previously (23 (link)–25 (link)). GFP-YVAD was PCR-amplified from pEGFP-C3 and cloned into the Lamp1-RFP plasmid (a gift from Walter Mothes; RRID: Addgene Cat. # 1817) (26 (link)) as an EcoRI [New England Biolabs (NEB); Cat. # R0101S]/BamHI (NEB; Cat. # R0136S) fragment to generate GFP-YVAD-RFP (Supplementary Figure S1). Ligation was performed using T4 DNA ligase (Promega; Cat. # M1801) using the protocol of the manufacturer for staggered end ligation. Caspase-1 mutants (Supplementary Figure S2A) were generated by QuikChange PCR. The parental caspase-1 plasmid (methylated) was digested using DpnI (NEB; Cat. # R0176S). Plasmids were transformed in Escherichia coli DH5α-competent cells (NEB; Cat. # C2987I). All site-directed changes were confirmed by sequencing. Oligonucleotide primers used in this project were produced by Integrated DNA Technology and are listed in Table 1. DNA transfections were carried out using FuGENE6 (2.5 μl/μg DNA) (Roche Applied Science; Cat. # 11988387001) as per the protocol of the manufacturer.
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