Figure EV5. CD22 colocalization with BCR and ligand‐dependent BCR signalling in primary Cd22 ^R130E B cells.

• A–C
  Dual‐colour SIM analysis of CD22, IgM and IgD. Cells were fixed, stained with Atto633‐ or ‐488‐conjugated antibody against CD22, IgM or IgD and settled onto non‐stimulatory coverslips. Cells were then embedded in agarose, imaged with SIM and analysed. (A) SIM images of CD22, IgM and IgD. (B) Cross‐correlation function. (C) Pearson correlation coefficient. Bars and numbers indicate the mean. Data are from three independent experiments. *P < 0.05 (Student's t‐test).
• D
  Wild‐type and Cd22 ^R130E primary B cells were treated with 5 μg/ml anti‐kappa, and intracellular calcium flux was measured by flow cytometry.
• E, F
  Wild‐type and Cd22 ^R130E primary B cells were stimulated with 5 μg/ml anti‐kappa for the indicated times. Cells were lysed and analysed by SDS–PAGE followed by immunoblotting with phospho‐CD19 (p‐CD19), phospho‐Akt (p‐Akt), phospho‐ERK (p‐ERK) and actin as a loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the unstimulated sample of the wild‐type cells, set as 1. Data are representative of two independent experiments.

Source data are available online for this figure.