Basal Glutathionylation of Na,K-ATPase α-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis

Figure 6

S-glutathionylation of α1-subunit of Na,K-ATPase after acute and prolonged hypoxia before and after TCEP (25 mM) treatment. SC-1 cells were grown either for 3.5 h ((a), (b)) or for 96 h (c), each at 20% and 0.2% pO₂. Cell lysates were incubated with ((b), (c)) or without (a) 25 mM TCEP during 30 min at 37°C and α1-subunit of Na,K-ATPase was detected by immunoblotting (IB). (d) α1-Subunit of Na,K-ATPase was immunoprecipitated (IP) from cell lysates by anti-α1 antibodies and glutathionylation was detected with anti-glutathione (anti-GS) antibodies. The original immunoblotting readouts are presented above. Bars represent changes in the S-glutathionylated (GSS-α/α) form of the protein normalized to its total amount. , mean ± SD. ((e), (f)) Immunoblots of Na,K-ATPase α1-subunit after immunoprecipitation with anti-α1 antibodies and TCEP (25 mM) treatment in denaturing conditions (8 M urea, 8% SDS). (e) Detection of total α1-subunit using anti-α1 antibodies. (f) Detection of glutathionylated α1-subunit using anti-GS antibodies.

(a)

(b)

(c)

(d)

(e)

(f)