ATAT1 regulates the formation of actin foci and organization of the BCR at the IS. Representative images of control (siCTL) or ATAT1-silenced (siATAT1) B cells seeded on glass coverslips containing BCR⁺ ligands for different time points and stained for actin (grayscale). Quantification of actin foci at each time point shows a significant reduction in siATAT1 cells at 30 min (bottom panel). (B) Top: Representative images of control and ATAT1-silenced B cells seeded on glass coverslips containing BCR⁺ ligands activated for 30 min, stained for actin (red) and GEF-H1 (green). Lower panels: Z-scan fluorescence profile of GEF-H1 (left) and quantification of actin-GEF-H1 overlap revealing a significant reduction upon ATAT1 silencing (right). (C) Top: Western blot analysis of phosphorylated FAK (pFAK) at 5 and 30 min in siCTL and siATAT1 B cells. Bottom: Quantification shows a significant reduction in pFAK levels at 30 min in ATAT1-silenced cell. (D) Representative images of B cells stained for actin (red), lysosomes (LAMP1, blue), and the BCR ( green) in siCTL and siATAT1 B cells activated for 30 min on glass coverslips containing BCR⁺ ligands. Graphs show quantification of BCR-actin and BCR-LAMP1 colocalization of images from D. Data consider n ≥ 30 cells pooled from N = 3 independent experiments. Scale bars are 10 μm. Inset scale bars are 2 μm. (A and C): Two-way ANOVA with Sidak’s multiple comparison test. (B and D): t test. P values illustrated with asterisks are * <0.05, ** <0.01, *** <0.001, and **** <0.0001. Error bars are mean ± SEM. Source data are available for this figure: SourceData F5.