Figure 4.

The absence of Myo1g decreases the amount of CD44 and lipid rafts on the surface of B cells. (A) Expression of CD44 in LPS plus IL4-activated B cells from WT or Myo1g-deficient mice. The graph shows the expression of CD44 in B lymphocytes (10,000 events in a gate of B220 + B cells). (B) Expression of CD44 in resting or activated B cells, one-way ANOVA test was used in these experiments, values are mean ± SD (***P < 0.001) (n = 4). (C) Binding of hyaluronic acid (HA-FITC) to resting or LPS plus IL4-activated B lymphocytes from WT or Myo1g-deficient B lymphocytes. HA binding was blocked using the IM7 mAb. One-way ANOVA test was used in these experiments, values are mean ± SD (***P < 0.001) (n = 7). (D) Immunodetection of CD44 in LPS plus IL4-activated B lymphocytes; α-CD44 (clone KM201) was used for western blotting. (E) Confocal images of LPS plus IL4-activated WT or Myo1g^−/− B cells. (F) The localization of CD44 as cytosol/plasma membrane index was calculated [mean fluorescence intensity (MFI) in the plasma membrane was divided by MFI in the cytosol] (~30 cells were counted per experiment, in three independent experiments, 100 cells per data set). Student’s t-test was used in these experiments, values are mean ± SD (***P < 0.001). (G) IgM or (H) GM1 in LPS plus IL4-activated B lymphocytes from WT or Myo1g-deficient B cells, the graphs show the expression of GM1 in B lymphocytes (10,000 events in a gate of B220 + B cells). (I) GM1 staining, one-way ANOVA test was used in these experiments, values are mean ± SD (*P < 0.05) (~60 cells were counted per experiment, in four independent experiments, 250 cells per data set). (J) Confocal images of permeabilized LPS plus IL4-activated WT or Myo1g^−/− B cells. Arrows indicate the localization of GM1 in B lymphocytes (scale bar 5 µm). (K) Intracellular versus plasma membrane GM1 (~30 cells were counted per experiment, in 3 independent experiments, 100 cells per data set), Student’s t-test was used in these experiments, values are mean ± SD (***P < 0.001).