Escherichia coli transetta de3

Recombinant GSDMEs and CASPs are all soluble and were purified as described previously (Jiang et al., 2020 (link); Xu et al., 2022 (link)). Briefly, the CDSs of TrGSDME and CASP variants were each cloned into pET30a (+), and the CDSs of HsGSDME, MmGSDME, and chimeric GSDME were each cloned into pET28a-SUMO. The recombinant plasmids were introduced into Escherichia coli Transetta (DE3) (TransGen, Beijing, China) by transformation. The transformants were cultured in Luria broth (LB) at 37°C until logarithmic growth phase. Isopropyl-β-d-thiogalactopyranoside (0.3 mM) was added to the medium, followed by incubation at 16°C for 20 hr. Bacteria were harvested and lysed, and the supernatant was collected for protein purification with Ni-NTA columns (GE Healthcare, Uppsala, Sweden). The proteins were dialyzed with PBS at 4°C and concentrated.

In vitro pull-down and competitive pull-down assays were carried out as previously described [57 (link)]. Constructs pMAL-p2x-OsIAA10, pMAL-p2x-OsIAA10P116L, pMAL-p2x-OsTIR1, pMAL-p2x-P2(1–786), pGEX-4T-1-OsIAA10, and pCST-P2(1–786) constructs, as well as empty pMAL-p2x and pGEX-4T-1 vectors were individually transformed into Escherichia coli Transetta (DE3) (Transgene; Beijing, China). Protein expression was induced by isopropyl-β-D-thiogalactoside (IPTG). Soluble MBP fusion proteins were extracted and immobilized onto amylose resin (New England Biolabs; Ipswich, MA, USA). Soluble GST fusion proteins were extracted and immobilized onto glutathione sepharose beads (GE Healthcare; Little Chalfont, Buckinghamshire, UK). For competitive pull-down assays, 3 μg of GST-OsIAA10 with 0, 3, 6, or 12 μg GST-P2(1–786) or 6 μg of GST alone were incubated with immobilized MBP-OsTIR1 (6 μg) at 4°C for 1 h. Proteins retained on the beads were resolved by SDS-PAGE and detected with anti-GST or anti-MBP antibodies, respectively.