Figure 4. Activation of calpain I in RBC_VL or RBC_N.

A. Cells (2×10⁷) were incubated with or without anti-9-O-AcSGP IgG_VL, anti-9-O-AcSGP IgG_NHS (10 µg/ml) or the Ca²⁺ ionophore A23187 in the absence or presence of EGTA (25 mM) for 60 min at 37°C in Ringer solution, washed with same buffer and lysed by sonication on ice. After removal of cell debris by centrifugation the lysates were separated by SDS-PAGE, and calpain I was detected by Western blot analysis with an anti-calpain-I antibody. B. Enhanced degradation of α-spectrin in sensitized RBC_VL. RBC_VL and RBC_N were suspended separately in Ringer solution and sensitized with anti-9-O-AcSGP IgG_VL, anti-9-O-AcSGP IgG_NHS or A23187 in the absence or presence of EGTA for 30 min at 37°C. The cells were then lysed as before and the lysates subjected to SDS-PAGE and Western blot analysis with a spectrin-specific antibody. C. Involvement of calpain I in the degradation of spectrin in RBC_VL. RBC_VL and RBC_N were pre-incubated with the calpain I inhibitor ALLN in Ringer solution for 30 min at 37°C before sensitization or co-incubated together with anti-9-O-AcSGP IgG_VL or A23187 as positive controls. The cells were processed as before and spectrin detected by Western blot after SDS-PAGE of the cellulaproteins. D. Dependence of the hemolysis of RBC_N on the activation of calpain I. RBC_N were incubated without or with A23187 and with A23187 plus EGTA or ALLN for 1 h at 37°C, and the degree of hemolysis determined spetrophotometrically as in Figure 1. The results are shown as representative bar graphs of three independent experiments.