Erythrophagocytosis Assay.

a<p>RBC_VL and RBC_N (2×10⁶), without or with sensitization by anti-<i>O</i>-AcSGP IgG_VL and anti-<i>O</i>-AcSGP IgG_NHS antibodies and processed for erythrophagocytosis assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042361#s2" target="_blank">Materials and Methods</a>. Results represent the mean ± S.D. of five separate determinations.</p>b<p>Positive macrophages (%) are the percentage of macrophages that ingested one or more erythrocytes and was used as the index of phagocytosis. Results represent the mean ± S.D. of five separate determinations.</p

Degradation of purified α-spectrin by activated calpain I.

<p><b>A... </b><i>Characterization of purified spectrins</i>. Spectrin_VL and spectrin_N were purified from RBC_VL and RBC_N as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042361#s2" target="_blank">Materials and Methods</a>, and analyzed on SDS-PAGE (7.5%). The purified spectrins were Western blotted and detected with polyclonal rabbit anti-spectrin antibodies. <b>B... </b><i>Proteolysis of purified spectrin_VL and spectrin_N by active calpain I</i>. Spectrin_N and Spectrin_VL (3.0 µg) were digested with different doses of active calpain I as indicated in reaction buffer, the reaction was stopped with EGTA and the products analyzed by SDS-PAGE. <b>C... </b><i>Inhibition of proteolysis by the calpain I inhibitor ALLN</i>. Calpain I was preincubated with ALLN for 15 min on ice prior to addition of reaction buffer containing purified spectrin_VL or spectrin_N and processed as before.</p

Activation of calpain I in RBC_VL or RBC_N.

<p><b>A.</b> Cells (2×10⁷) were incubated with or without anti-9-<i>O</i>-AcSGP IgG_VL, anti-9-<i>O</i>-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>B... </b><i>Enhanced degradation of α-spectrin in sensitized RBC_VL</i>. RBC_VL and RBC_N were suspended separately in Ringer solution and sensitized with anti-9-<i>O</i>-AcSGP IgG_VL, anti-9-<i>O</i>-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. <b>C... </b><i>Involvement of calpain I in the degradation of spectrin in RBC_VL</i>. 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-<i>O</i>-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. <b>D... </b><i>Dependence of the hemolysis of RBC_N on the activation of calpain I</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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042361#pone-0042361-g001" target="_blank">Figure 1</a>. The results are shown as representative bar graphs of three independent experiments.</p