Protein a resin

General co-IP procedure similar to that previously reported was followed^{63 (link)}. Briefly, HEK293T cells were treated with either DMSO (–) or HNE (+) (12 µM). After 1.5 h, cells were harvested and washed twice with PBS. Cells were resuspended in lysis buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton-X-100, 1X Roche cOmplete, EDTA-free protease inhibitor cocktail) and freeze-thawed three times. The lysate was spun down (20,000 × g, 10 min at 4 °C). The supernatant was collected and normalized to 12 mg/ml. 1 mL lysate was precleared with 150 μl settled protein A resin (ThermoFisher, #20334) at 4 °C for 1 h. To the 1 mL precleared lysate was added 75 μl mouse anti-Keap1 bound protein A resin and incubated at 4 °C overnight (the mouse anti-Keap1 bound protein A resin was produced by incubating 1 μg mouse anti-Keap1 with 100 μL settled protein A resin at 4 °C overnight a day prior to the experiment). The resin was pelleted (1000 × g, 1 min) then washed three times for 10 min each in 1 ml of 50 mM Tris pH 7.6, 150 mM NaCl, 0.1% Triton X-100 at 4 °C. The washed resin was added 50 μl 2X Laemmli Buffer containing 50 mM TCEP, and heated to 95 °C for 5 min to elute proteins. The resin was spun down (20,000 × g, 10 min at rt), and the supernatant was used in SDS-PAGE and western blot analyses.

PGT125, 126 128 and 130 Fabs were expressed in FreeStyle 293S cells (Life Technologies) grown in suspension by transfection at a 1:1 ratio of plasmids encoding light and heavy chain (truncated at Asp^H234). Supernatants harvested 6 days after transfection were passed over an anti-human lambda affinity matrix (CaptureSelect LC-lambda (hu); Thermo Scientific) equilibrated in PBS. Fab fragments were eluted with 0.1 M glycine, pH 3.0 and neutralized with 10 × concentrated PBS. Recombinant IgGs of all antibodies used in this study were expressed in FreeStyle 293 F cells and purified on a protein A resin (Thermo Scientific) using the aforementioned glycine solution for elution, as recently described^{20 (link)}.