Efficient silencing of ATAT1 in B cells, followed by rescue of its expression, restores tubulin acetylation, whereas leptomycin-treated B cells display reduced tubulin acetylation. (A) Left: Representative confocal images of control (siCtl) or ATAT1-silenced (siATAT1) B cells transfected, activated on stiff or soft substrates, and stained for ATAT1 (green). White outlines indicate cell contours, obtained by F-actin staining. Right: Line-scan profiles of ATAT1 fluorescence intensity across the cell diameter. (B) Quantification of ATAT1 fluorescence intensity from cells shown in A. (C) Representative confocal images of siATAT1 B cells expressing ATAT1-venus, empty vector, or non-transfected cells, activated on glass slides coated with BCR⁺ ligands for 5 and 10 min. (D) Quantification of Ac-Tub fluorescence intensity from cells shown in C. (E) Western blot of Ac-Tub and α-Tub (loading control) in B cells activated for 0, 5, 15, or 30 min in the presence of vehicle (EtOH) or LMB. Shown data consider n ≥ 30 cells pooled from N = 3 independent experiments. Scale bars are 10 μm. Two-way ANOVA with Sidak’s multiple comparison test. P values illustrated with asterisks are ** <0.01 and *** <0.001. Source data are available for this figure: SourceData FS4.