Ab19534

Recombinant Ig-domain proteins were incubated with 2 mM GSH and 0.5 mM diamide for 30 min at 27 °C, 57 °C, or 77 °C in a shaker; 5 mM DTT was also used to reverse oxidation. Proteins were analyzed by immunoblotting using anti-GSH antibody (ab19534, Abcam; 1:2,000). Phosphorylation by CaMKIIδ (Merck Millipore) was detected by autoradiography.

Cells were lysed using NP40 cell lysis buffer (Beyotime). The lysate obtained was then subjected to centrifugation and subsequently incubated with antibodies specific to Keap1 or Flag (no. 14793, CST, Danvers, MA, USA) overnight at 4 °C. Protein G magnetic beads (ThermoFisher) were used to purify the immune complexes at 4 °C for 2 h, followed by centrifugation and washing with NP40 cell lysis buffer for 4–5 times. The immunoprecipitated protein was further analyzed by western blotting using an anti-Nrf2 or anti-GSH antibody (ab19534, Abcam, Cambridge, UK).
For the determination of S-glutathionylated Keap1, cells were lysed in ice-cold lysis buffer supplemented with 10 mM NEM. The supernatants of the whole-cell lysates were incubated with an antibody specific to GSH, subsequently purified by Protein G magnetic beads, and then resolved by SDS-PAGE under non-reducing conditions, followed by incubation with a Keap1 antibody.

Blood samples collected from 12 study participants at sea level and high altitude (see Fig. 1A and [22 ] for more details). Washed RBC pellet was snap-frozen in liquid nitrogen and stored at −80 °C. Samples were thawed on ice and supplemented with a protease inhibitor cocktail (1:200) and 25 mM NEM in PBS to prevent oxidation of SH-groups. Negative control was prepared by treatment of a sample with 100 mM dithiothreitol. Primary mouse Anti-Glutathione antibody (ab19534, abcam 1:1000) were used for detection of S-glutathionylated proteins and rabbit anti-human Recombinant Anti-Hemoglobin subunit beta/ba1 antibody (ab214049, Abcam 1:1000) for detection of Hb. Densitometry was performed using Image studio lite (LI-COR Biosciences) and the signal for GSH was normalized to that for the βHb monomer. Finally, the readouts obtained for different time points were normalized to the basal level of glutathionylation (time-zero for ex vivo experiments or samples before the HA exposure). Similar procedure was performed with RBC hemolysates obtained from RBC suspensions exposed to air or pure N₂ or air in Eschweiler tonometers at 37 °C.