Cdc42 is essential to establish the mature B cell compartment. (A) Splenic WT and Cdc42 KO B cells were isolated and protein expression levels of Cdc42 (top row) and actin (bottom row) were analyzed by Western blot. Densitometric analysis was used to quantify the signal intensity of Cdc42 normalized to actin and is shown in the chart below. (B and C) Spleen (B) and inguinal LNs (C) from WT and Cdc42 KO mice were analyzed by flow cytometry. Gated populations are as follows: B cells (first row in B and C, B220⁺), marginal zone B cells (second row, B220⁺CD21^hiCD23^low), and double negative B cells (third row, B220⁺CD21⁻CD24⁻). Quantification charts are shown in the right-hand column and represent percentage of live cells (first row in B and C) or percentage of B220⁺ cells (second and third rows in B). (D–E) Frozen sections from spleens (D) or inguinal LNs (E) from WT and Cdc42 KO mice were stained with antibodies against B220, TCRβ, and F4/80 (D) or CD169 (E), and tiled images were acquired with a confocal microscope. Bars, 800 µm. Data were pooled from 3 experiments with n = 8 WT mice and n = 4 Cdc42 KO mice. Mean and SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.0001.