BCR-mediated spreading is reduced in human B cells lacking INPP5B. (A) Protein extracts from WT Ramos and INPP5B^KO/KO cells were subjected to IP using sheep anti-INPP5B antibody. INPP5B was detected by Western blotting using a rabbit anti-INPP5B. (B) Analysis of BCR surface expression in WT vs. INPP5B-KO clones by flow cytometry. (C) Representative TIRF microscopy images from WT vs. INPP5B-KO cells that were settled on coverslips presenting surrogate antigens for the indicated time points. F-actin was stained with Phalloidin. Scale bar, 10 μm. Statistical analyses of cell spreading area (at 5 and 15 min) are shown on the right. The data are pooled from three independent experiments. Error bars indicate SD, and P values were calculated using Tukey’s multiple comparisons test. (D) Representative TIRF microscopy images from DMSO (vehicle)- vs. INPP5B inhibitor (YU142670)–treated WT and INPP5B-KO clone 2 cells settled on coverslips presenting surrogate antigens. Analysis of cell spreading was performed as in C, and data on the right were pooled from three independent experiments. (E) Freshly isolated untouched human primary B cells were treated with DMSO (vehicle) vs. INPP5B inhibitor for 30 min, before activation on coverslips presenting surrogate antigens. Cell spreading was analyzed at 15 min, and data on the right were pooled from four independent experiments (using PBMCs isolated from three different healthy donors). The P value was calculated using Wilcoxon matched-pairs signed rank test. (F) Human primary B cells treated with DMSO vs. Cdc42 inhibitor (ML141) vs. Arp2/3 inhibitor (CK666) were settled on antibody-coated coverslips and analyzed as previously. The data represent mean ± SD of 45 cells, and P values were calculated using Dunnett’s T3 multiple comparisons test. Source data are available for this figure: SourceData F8.