Fig. 5: Dominant C3 activation in splenic lipid rafts in a SIGN-R1-dependent manner following S. pneumoniae challenge in vivo.

a Spleens, livers, and lungs of control mice were fractionated with sucrose gradient ultracentrifugation, and fractions were immunoblotted for C3. b As in a, but wild-type mice were injected intravenously with PBS or live S. pneumoniae (Pn14; 1 × 10⁸, 1 h). c As in a, b, but SIGN-R1-KO mice were used, and splenic fractions were analyzed. d, e As in c, but mice were injected intravenously with an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus (1 × 10⁸, 1 h). f As in c, but CVF-treated mice were used, and splenic fractions were immunoblotted for C3, SIGN-R1, C1q, and C4. g DCEK_WT and DCEK_SIGN-R1 were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10⁷, 1 h) without or with 5% NMS in TC buffer for 15 min at 37 °C. Cells were immunostained for C4 or C3 with their respective isotype control IgGs and assessed by FACS. h As in g, but the binding of C4 (red) or C3 (green) was assessed by fluorescence microscopy after fixing DCEK-SIGN-R1 transfectants with 1% paraformaldehyde. Arrowheads indicate colocalization of C4 and C3. Representative cells are shown in the box. All data are representative of five independent experiments. Scale bars h, 10 µm