BCR cluster formation, B-cell spreading, and BCR signalosomes are reduced in DKO B cells. Splenic B cells were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37°C for indicated times (n = 4). Cells were fixed, permeabilized, and stained for pCD19 and pY. Shown are representative images (A-D) and the average values (± standard deviation) of the MFI of the BCR (E), B-cell contact area (F), the MFI of pY (G), and the MFI of pCD19 (H) in the contact zone. The data were generated by using 20 to 90 cells from 3 independent experiments. Scale bars, 2.5 μm. One-way analysis of variance with the Tukey test was used to perform multiple group comparisons; *P < .01.