Accumulation of PI(4,5)P ₂ in INPP5B-depleted cells results in defective BCR clustering. (A) Two-color time-lapse TIRF microscopy images of PI(4,5)P₂ (EGFP-Tubby) and BCR in vehicle-treated INPP5B^{Degron/Degron} cells that were stimulated on antibody-coated glass. Images are pseudo-colored. Scale bar, 10 μm. (B) Representative images of PI(4,5)P₂ and BCR in auxin-treated INPP5B^{Degron/Degron} cells stimulated on glass as above. Boxed areas are magnified in time sequence (bottom rows). Scale bar, 1 μm. (C) Normalized MFI of PI(4,5)P₂ (EGFP-Tubby) foci over time starting 2 min after contact with the antibody-coated coverslip. n = 20 foci; error bars indicate SD. (D) Normalized MFI of BCR staining over time at the same membrane contact regions in C. (E) Normalized MFI ratio of PI(4,5)P₂/BCR over time. Data pooled from three independent experiments were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. n = 20 foci (regions of PI(4,5)P₂ enrichment); error bars indicate SD.