Fig. 3: Accumulation and multimerization of SIGN-R1 in splenic lipid rafts following exposure to CPS14 from S. pneumoniae in vivo.
a DCEK_SIGN-R1 transfectants were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 106, 10 min) at 37 °C or 4 °C and immunostained for SIGN-R1 without permeabilization. b As in a, but cells were pretreated with MβCD (10 mM, 3 h) and incubated only at 37 °C. c As in a, but cell lysates at 37 °C were fractionated with sucrose gradient ultracentrifugation, and fractions of LRs were immunoblotted for SIGN-R1, flotilin-1, or caveolin-1. Multimers of SIGN-R1 are shown in the boxes. d In total, 1 × 108 CFSE-labeled MitC-Pn14 (green) were injected intravenously into wild-type mice for 0, 15, or 60 min, and splenic sections were immunostained for SIGN-R1 (blue). The binding or uptake of organisms into splenic MZs is shown in the boxes. (E) As in (C), but spleens were used before or after intravenous injection of live S. pneumoniae (Pn14; 1 × 108, 1 hr) into wild-type mice. (F and G) As in e, but mice were injected intravenously with PBS or 1 × 108 cells of an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus for 1 h, respectively. h As in e, but fractions were immunoblotted for MARCO, SER4/CD169, flotilin-1, and caveolin-1. Scale bars a, b, 20 µm; d, 250 µm
