4.2.1. Treatment of Erythrocytes

RBCs were pelleted at 3750× *g* for 3 min. After removal of the supernatant, packed RBCs were washed three times at 3750× *g* for 3 min in 5 volumes of Dulbecco's Phosphate Buffered Saline (D-PBS) to avoid contamination by leucocytes and platelets. Packed cells (50 µL) were suspended (at 20% hematocrit) in D-PBS and treated at 35 ◦C for 30 min in the absence (Basal) or presence of 1.5 mM diamide (dissolved in D-PBS) (Diamide), or 0.3 mM DDS-NHOH dissolved in acetone (DDS-NHOH).

After washing, RBCs underwent hemolysis in 1.5 mL of hypotonic buffer (5 mM sodium phosphate, pH 8, 0.02 % sodium azide (NaN3), 30 µM phenylmethylsulphonyl fluoride (PMSF), 1 mM sodium orthovanadate, and a protease inhibitor cocktail) [24].

Membranes were separated from the cytosol by centrifugation (16,100× *g* for 20 min in an Eppendorf centrifuge) and washed once in a hypotonic buffer. Aliquots of membranes and the cytosol were analyzed by Western blotting in reducing or non-reducing conditions and immunostained with appropriate antibodies.
