Supplementary Figure 2: TLR9 signaling does not affect the internalization dynamics of BCR bound soluble antigen.

The internalization of BCR bound soluble antigen was quantified in splenic B cells purified from WT or MD4 mice. (a) The percent internalization of Alexa-647-labeled Anti-IgM by WT B cells in the presence or absence of CpG was measured with time at 37 °C as detailed in Methods. (b) The internalization of HEL by MD4 HEL-specific B cells was determined by measuring the decrease in surface BCR over time at 37 °C as described in the Methods section. Data are representative of three independent experiments each of which were performed in duplicates. (c-g) To visualize the cytoplasmic compartments containing internalized antigen, purified B cells were incubated with Miltenyi metal particles coated with Anti-IgM in the presence or absence of 1µM CpG. Cells were fixed at 60 min and analyzed later using tomographic TEM. Representative reconstructed images are shown (c). White arrows indicate vesicles containing internalized particles. N marks the nucleus. Sections taken from 12 different cells per group were used to quantify the number of bead-containing vesicles in each section (d), the number of beads per each vesicle (e), volume of the bead-containing vesicles (f) and number of beads per vesicular volume (g). Lines indicate mean values. Statistical significance was measured by two sided unpaired t-test.