The expression of EGFP-Tubby does not perturb BCR clustering or signaling. (A) PIP₂ levels in INPP5B-depleted cells. INPP5B^{Degron/Degron} cells were stimulated in solution for the times indicated, before analysis by MS. PIP₂ levels are expressed relative to PI, and data for the C40:6, C36:2, C38:2, and C38:4 species or a pool of all four are shown separately. Data from three independent experiments were analyzed by two-way ANOVA. (B) Representative TIRF microscopy images from parental INPP5B^{Degron/Degron} cells or a derivative clone expressing EGFP-Tubby that were settled on coverslips presenting surrogate antigens for 15 min. F-actin was stained with Alexa Fluor 488–Phalloidin. Scale bar, 10 μm. Statistical analyses of the spread area and the MFI of BCR staining at the contact interface are shown on the right. The data represent mean ± SD of 22 cells. (C) Western blot analysis of phospho-ERK in lysates from INPP5B^{Degron/Degron} cells stably expressing EGFP-Tubby. The parental cells were used as a control. The quantification of blots (from a single experiment) is shown on the right. (D) Representative TIRF microscopy images at the indicated time points of INPP5B^{Degron/Degron} cells expressing a fluorescent biosensor (tubby) after stimulation on antibody-coated coverslips. Scale bar, 10 μm. Images are pseudo-colored. See Videos 3 and 4 for the complete time-lapse TIRF microscopy images. Analysis of the intensity of PI(4,5)P₂ per area (MFI; top) and the spreading area (bottom) at the indicated time points in vehicle- vs. auxin-treated cells (n = 20) are shown on the right. Data was analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. Bars represent mean ± SD. Source data are available for this figure: SourceData FS3.