F-actin turnover at the cell-coverslip contact interface is required for BCR clustering. (A) Two-color time-lapse TIRF microscopy images of F-actin (LifeAct-EGFP) and BCR in vehicle-treated (top rows) and auxin-treated (bottom rows) INPP5B^{Degron/Degron} cells that were stimulated on antibody-coated glass. Images are pseudo-colored. (B) Representative TIRF microscopy images showing BCR and F-actin (LifeAct-EGFP) of vehicle- vs. auxin-treated INPP5B^{Degron/Degron} cells at 5 min. Fluorescence intensity profiles of F-actin and BCR on the white line in the TIRF microscopy images are shown on the right. (C) GTP-bound Rap1 was detected using RalGDS-RBD GST pulldown followed by blotting against Rap1. Quantification of two such experiments by densitometry is shown below. GTP levels of Rap1 are expressed relative to total Rap1. Source data are available for this figure: SourceData FS4.