The inositol 5-phosphatase OCRL is not required for BCR clustering or signaling. (A) Cell-free extracts from WT DT40 cells and OCRLDegron/Degron cells that were treated with or without auxin for 2 h were blotted with an anti-OCRL antibody. Analysis of three such blots are shown on the right. (B) Representative confocal images at the indicated time points from OCRLDegron/Degron cells stimulated with Texas Red–conjugated anti-IgM antibody. Analysis of BCR capping is shown on the right. Scale bar, 10 μm. (C) Representative TIRF microscopy images from BCR-labeled OCRLDegron/Degron cells that were settled on coverslips presenting surrogate antigens for 15 min. F-actin was stained with Phalloidin. Scale bar, 5 μm. (D–F) Statistical analyses of TFI (BCR), spread area, and MFI (BCR) from vehicle-treated and auxin-treated OCRLDegron/Degron cells in D, E, and F, respectively. The data represent mean ± SD of 15 cells. (G) Protein extracts from OCRLDegron/Degron cells stimulated in solution were blotted for Akt and ERK phosphorylation at the indicated time points. (H) Quantification of the blots (from a single experiment) by densitometry. (I) Protein extracts from OCRLDegron/Degron cells stimulated on glass coverslips were blotted for Akt and ERK phosphorylation. (J) Quantification of the blots (from a single experiment) by densitometry. Source data are available for this figure: SourceData FS2.
