Figure 5.

IgG1-BCRs rapidly undergo BCR oligomerization-induced changes in the BCR’s cytoplasmic domains and recruit pSyk. (A–C) FRET efficiencies between FRET donor Igα-YFP and FRET acceptor IgH-CFP are given at the indicated times for J558L cells placed on lipid bilayers containing NIP-H12. Acquisition and analyses of CFP and YFP paired TIRF FRET images were as reported (Tolar et al., 2005) and detailed in the Methods section. (A) Mean ± SD of FRET efficiencies are given over 240 s for γ1-High and μ-High. (B) Statistical comparisons for the maximal changes in FRET (ΔFRET) from 0 and 200 s are given. The decay plots of FRET efficiencies from maximal FRET values, set at 100%, are given. (C) The decay plots were mathematically fitted into a mono-exponential decay function to calculate the half life of FRET loss, τ₅₀, as detailed in the Methods section. (D, E) The γ1-High and μ-High J558L cells were placed on lipid bilayers containing NIP-H12 for 10 min, fixed, permeabilized and stained with antibodies specific for pSyk as detailed in Methods section. (D) Given are two-color TIRF images for Igα-YFP (green) and pSyk (red). For the original 16-bit TIRF images, the display range was set from 928 to 1344 (Igα-YFP) and from 928 to 992 (pSyk) in both γ1-High and μ-High cells to allow direct visional comparison. Scale bar is 1.5μm. (E) Given are the number of pSyk clusters in the contact area, the size of the contact area and mean BCR FI of the contact area for γ1-High and μ-High cells. Each dot represents one cell analyzed in three independent experiments and bars represent the mean ± SD. Two-tailed T tests were performed for the statistical comparisons. The results given for μ-High J558L cells were recently reported (Liu et al., 2010). See also Figure S5.