Figure 1. BCR signaling induces transient dephosphorylation of ERM proteins. (A and B) Primary WT splenic B cells were incubated on glass slides coated or not with 10 µg/ml anti-κ antibody for the indicated periods of time and then fixed and stained for threonine phosphorylation of ERM proteins (pERM) and imaged by confocal microscopy. (A) Representative image. (B) Quantification of the change in mean fluorescence intensity (MFI) of pERM in individual cells over time upon stimulation. Red bars indicate mean ± SEM. *, P = 0.0198; ***, P < 0.0001. (C–E) Primary WT (C, top), PLCγ2-KO (C, middle), or Vav1/2-KO (C, bottom) B cells labeled with Cy3 anti-IgM Fab were fixed on antigen-containing lipid bilayers after 1.5 min of interaction for subsequent pERM immunofluorescence and imaged by TIRFM. (C, left) Representative images. (right) Fluorescence intensity profile of BCR and pERM along the white lines. (D) Quantification of the segregation of BCR and pERM fluorescence in individual cells using Pearson’s correlation coefficient, with the mean ± SEM indicated by the red bars. *, P = 0.0211; ***, P < 0.0001. (E) Quantification of mean pERM fluorescence intensity at the cell–bilayer contact, with the mean ± SEM indicated by the red bars. ***, P = 0.0008 (PLCγ2-KO); ***, P < 0.0001 (Vav1/2-KO). Data are representative of two independent experiments. Bars, 2 µm.
Figure 1.

BCR signaling induces transient dephosphorylation of ERM proteins. (A and B) Primary WT splenic B cells were incubated on glass slides coated or not with 10 µg/ml anti-κ antibody for the indicated periods of time and then fixed and stained for threonine phosphorylation of ERM proteins (pERM) and imaged by confocal microscopy. (A) Representative image. (B) Quantification of the change in mean fluorescence intensity (MFI) of pERM in individual cells over time upon stimulation. Red bars indicate mean ± SEM. *, P = 0.0198; ***, P < 0.0001. (C–E) Primary WT (C, top), PLCγ2-KO (C, middle), or Vav1/2-KO (C, bottom) B cells labeled with Cy3 anti-IgM Fab were fixed on antigen-containing lipid bilayers after 1.5 min of interaction for subsequent pERM immunofluorescence and imaged by TIRFM. (C, left) Representative images. (right) Fluorescence intensity profile of BCR and pERM along the white lines. (D) Quantification of the segregation of BCR and pERM fluorescence in individual cells using Pearson’s correlation coefficient, with the mean ± SEM indicated by the red bars. *, P = 0.0211; ***, P < 0.0001. (E) Quantification of mean pERM fluorescence intensity at the cell–bilayer contact, with the mean ± SEM indicated by the red bars. ***, P = 0.0008 (PLCγ2-KO); ***, P < 0.0001 (Vav1/2-KO). Data are representative of two independent experiments. Bars, 2 µm.

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