NEMO Protein Expression and Purification

NEMO(1–120) was subcloned into the pDEST-His-SUMO vector containing an N-terminal His₆-SUMO tag (Invitrogen) as previously described^{28 (link)}. Wild-type NEMO (WT-NEMO), 5XAla-NEMO, and 7XAla-NEMO were subcloned as described previously ^{19 (link),28 (link)} into the pET24b(+) vector (Novagen) via NdeI and XhoI sites, with WT-NEMO transformed into Rosetta 2(DE3)pLysS competent cells (Novagen) and 5XAla-NEMO and 7XAla-NEMO transformed into T7 Express I^q Competent E. coli cells (New England Biolabs). NEMO(44–195) and NEMO(110–195) were subcloned out of a codon-optimized template (GenScript) by polymerase chain reaction, and ligated into a SUMOstar vector (LifeSensors) via BsaI and XhoI sites. These constructs were transformed into T7 Express competent cells (New England Biolabs) to take advantage of the codon optimization. 9SG was subcloned from the native, non-codon optimized 7XAla-NEMO template using the NEB Q5 Site-Directed Mutagenesis Kit (New England Biolabs), and transformed into Rosetta 2(DE3)pLysS competent cells (Novagen).