Tandem affinity purification of KEAP1 complexes

Generation of the HeLa S3 cell line stably expressing FLAG-HA double tagged KEAP1 and tandem affinity purification of the KEAP1 protein complexes were carried out following previously described procedures (22), with modifications mostly to fit smaller scales. Briefly, the cell lines were generated by transducing cells with the bicistronic retroviral vector pOZ-FH-C-KEAP1 (11), which expresses C-terminally tagged KEAP1 from the first cistron and the interleukin receptor α (IL2α) from the second, followed by selection with the paramagnetic Dynabeads® Goat Anti-Mouse IgG (Invitrogen) coupled with an anti-interleukin 2 receptor α (IL2α) antibody (clone 7G7/B6, Upstate). KEAP1 complexes were purified from ∼2 × 10⁸ cells under each condition. Cytoplasmic and nuclear contents were separated by hypotonic swelling and douncing, and the respective extracts were prepared in NETNG250 (20 mM Tris-HCl [pH7.5], 250 mM NaCl, 0.5% NP-40, 2 mM EDTA, 10% glycerol) with the Complete® protease inhibitor cocktail (Roche). FLAG-HA-tagged KEAP1 protein complexes were purified from cytoplasmic and nuclear extracts by two rounds of affinity purification using anti-FLAG M2 agarose (Sigma) and anti-HA agarose beads (Sigma). The final material bound to the anti-HA beads was eluted with 0.1 M glycine (pH 2.5) and neutralized with 0.1 volume of 1 M Tris-HCl (pH 8.5). Purified material was resolved on a 4-12% Tris-Glycine SDS gel (Invitrogen), and proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).