Fig. 2: Distribution of SIGN-R1 in splenic lipid rafts and its role in the uptake and decomposition of S. pneumoniae.

a (Left) DCEK_SIGN-R1 transfectants were immunostained for SIGN-R1 (red), cholera toxin b (green), and DAPI (blue). Arrowheads (yellow) indicate the colocalization of DC-SIGN and cholera toxin B. (Right) After sucrose gradient ultracentrifugation of whole cell lysates of DCEK_SIGN-R1 transfectants, fractions were immunoblotted for DC-SIGN, flotilin-1, or caveolin-1. Multimers of SIGN-R1 are presented in the box. b, c As in a (right), but spleens from wild-type and SIGN-R1-KO mice were used and immunoblotted for SIGN-R1, flotilin-1, and caveolin-1. d (Left) DCEK_SIGN-R1 transfectants were treated with MβCD (10 mM, 3 h), immunostained for SIGN-R1 without permeabilization, and assessed by FACS. (Right) As in (left), but cells were immunostained for cholera toxin B (green) and SIGN-R1 (red) and followed by microscopic analysis. e Cells in d were further incubated with FITC-dextran (5 μg, 30 min) or CPS14 (10 μg, 2 h), and their uptake was assessed by FACS (green or pink lines for uptake without or with MβCD, respectively). f–h (Left) DCEK_SIGN-R1 transfectants were pretreated with f MβCD, actinomycin-D (AD; 5 μg/mL, 24 h), cycloheximide (CD; 20 μg/mL, 24 h) or with g myristoylated dynamin inhibitory peptide (50 μM, 1 h)⁷¹ and washed out, or h transfected with dominant-negative dynamin (K44A) and incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10⁶, 15 h, 37 °C), followed by immunostaining for f, g SIGN-R1 (green) and CPS14 (red) or (H) dynamin (green) and CPS14 (red). (Right) The average percentage of pneumococcal decomposition of total pneumococcal binding on SIGN-R1 transfectants was calculated in five areas from each sample in five independent experiments. Data are shown as mean ± SD. n.s., Not significant; *p < 0.05; **p < 0.01; ***p < 0.001., Scale bars a, f, g, h, 20 µm; d, 10 µm