Effect of diamide exposure on RBC membrane protein–protein associations and deformability. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of dRBCs, run in nonreducing (A1) or reducing (A2) conditions. (B) Deformability profile of dRBCs or untreated control RBCs before the spleen perfusion (shown is a representative experiment, n = 8), as assessed by Lorrca. (C) EI (RBC deformability parameter) is plotted as a function of the logarithm of the shear stress. dRBCs required significantly greater applied shear stress to reach equivalent deformation as RBCs with normal membrane. Because the lines are parallel, one can see that RBC membranes treated with 10 µM diamide required 1.3-fold greater shear stress than normal membranes to reach equivalent deformation at all points along the curve, indicating that the diamide-treated membranes had 0.79 times the normal deformability. Treatment with 125, 300, and 500 mM diamide resulted in membranes that had 0.29-, 0.14-, and 0.07-fold the normal deformability, respectively. (D) Linear regression fit of the correlation between diamide concentrations and the relative rigidity of dRBCs. (E) Osmotic fragility of dRBCs (a representative experiment).