Figure 3.
WIP Is Required for Efficient BCR-Induced Activation of the PI3K Pathway
(A) TIRF microscopy of WEHI-231 B cells expressing GFP-WIP imaged after spreading over a planar lipid bilayer labeled with anti-κ-chain antibody linked to Alexa Fluor 633 (left) and quantification of fluorescent intensities along the white diagonal line (right, colors match labels above images). Scale bar, 3 μm.
(B) Flow cytometric analysis of WIP phosphorylation after anti-κ-chain stimulation of WT B cells. Grey shaded histogram represents the unstimulated WT control. Graph shows the mean fluorescent intensity (MFI) normalized to the MFI of Wipf1−/− cells at indicated time points.
(C) Phalloidin staining indicating intracellular amount of F-actin in fixed WT or Wipf1−/− B cells settled on coverslips coated with anti-κ-chain antibody for 10 min. Scale bar, 3 μm. Quantification (right graph) shows F-actin foci per cell (mean ± SEM). Data are representative of three independent experiments.
(D and E) Immunoblot of splenic WT or Wipf1−/− B cells stimulated with anti-κ-chain antibody and probed with antibodies as indicated. Quantifications of intensity of proteins normalized by densitometry to total Erk or tubulin and to the signal in unstimulated WT cells at t = 0. Data are representative of at least three independent experiments.
(F) Graph show mean (±SEM) percentage of live WT or Wipf1−/− B cells cultured in medium or in the presence of BAFF at different time points, normalized to the starting input number of live B cells set to 100%. Results pooled from three independent experiments with one mouse each.
(G) Immunoblot of splenic WT or Wipf1−/− B cells stimulated with BAFF, probed with antibodies against p-Akt and Erk. Quantifications of intensity of proteins normalized by densitometry to total Erk and to the signal in unstimulated WT cells at t = 0.
(D and G) Experiments were performed simultaneously with experiments in Figure 5A and therefore results can be directly compared. The loading control measurements (Erk for anti-κ-chain and BAFF stimulation) were part of both experiments.