Dithiothreitol (dtt)

Recombinant Cys64Ser Cys82Ser Gpx3 protein was expressed and purified as described previously^{[40 (link)]}. The protein was reduced with 10 mM DTT (Research Products International Corp) during for 1h at 4°C prior to the removal of small molecules on Nap5 columns (GE Healthcare). 25 μM of Gpx3 was labeled with 10 mM of the different probes (DMD, CPD or DMBA) in presence of 1.5 Eq H₂O₂ (Sigma-Aldrich) for 2 h at room temperature with constant shaking. 5 μg of the samples were subjected to SDS-PAGE, and separation was performed at 165 V for 35 min. The gel was Coomassie stained with Coomassie during 20 min and destained overnight. The gel band corresponding to the expected molecular size of Gpx3 was cut from the gel, treated with 10 mM DTT / 50 mM NEM (Acros Organics) and subjected to trypsin digestion as described previously^{[32 (link)]}.

1 nmol GST-tagged proteins (either GST-SNX27_FL or GST-SNX1_N-terminal (1-142) or SNX1_N-terminal (1-60 aa) or SNX1_N-terminal (1-70 aa) or SNX1_N-terminal (1-80 aa) or SNX1_N-terminal (1-90 aa) or SNX1_N-terminal (1-100 aa) or SNX1_N-terminal (40-60 aa) or SNX1_N-terminal (40-70 aa) or SNX1_N-terminal (40-80 aa) or SNX1_N-terminal (40-90 aa) or SNX1_N-terminal (40-100 aa) or GST-SNX2_N-terminal (1-139) were mixed with 1 nmol of His-tagged proteins (either SNX1_FL or SNX1_PX-BAR or SNX1_BAR or SNX27_FL or SNX27_PDZ or SNX27_PX or SNX27_FERM), for 1 hr at 4°C. Protein mixture was then centrifuged at high speed to remove any precipitated proteins. The supernatant was then added to glutathione Sepharose pre-equilibrated in 20 mM Tris (pH 8.0), 300 mM NaCl, 1 mM DTT (Research Products International) and allowed to mix for 30 minutes at 4°C. Beads were washed five times in the above buffer supplemented with 0.5% Triton-X100 (Sigma Aldrich). Bound proteins were analyzed by SDS-PAGE and Western blots using HRP conjugated mouse anti-His antibody (Abcam) with 1:8000 dilution.