The impact of INPP5B depletion on BCR clustering and signaling is dependent on the enzyme’s catalytic activity. (A) RT-PCR analysis of the WT and mutant clones using gene-specific primers flanking the inpp5b stop codon. (B) Analysis of BCR expression in mutant clones by flow cytometry. (C) Representative confocal images at the indicated time points from auxin-treated INPP5B^Degron/WT and INPP5B^Degron/G502D cells stimulated with Texas Red–conjugated anti-IgM antibody. Scale bar, 10 μm. Statistical comparison for the percentage of cells showing BCR capping in INPP5B^Degron/WT versus INPP5B^Degron/G502D is shown on the right. Error bar represents SD, and the P value was calculated using Welch’s t test. (D) Statistical comparison for the MFI of cell surface BCR from INPP5B^Degron/WT vs. INPP5B^Degron/G502D cells over time. Data were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. Error bars indicate SD. (E) Representative TIRF microscopy images from auxin-treated INPP5B^Degron/WT and INPP5B^Degron/G502D cells that were settled on coverslips presenting surrogate antigens for 15 min. F-actin was stained with Phalloidin. Scale bar, 10 μm. (F–H) Statistical analyses of the TFI of BCR staining, spread area, and the MFI of BCR at the contact interface are shown in F, G, and H, respectively. Data (n = 35 cells) were pooled from three independent experiments. Bars represent mean ± SD, and P values were calculated using Welch’s t test. (I and J) Protein extracts from auxin-treated INPP5B^Degron/WT vs. INPP5B^Degron/G502D cells stimulated in solution (I) or on glass coverslips (J) were blotted for Akt and ERK phosphorylation at the indicated time points. Data from three independent experiments were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. Error bars indicate SEM. Source data are available for this figure: SourceData F3.