Rab43is highly and selectively expressed in CD8α⁺ cDCs and does not impact cDC development. (A) Sorted WT CD8α⁺ DCs, CD8α⁻ DCs, and BM CDPs analyzed by gene expression microarray. Expression levels were determined for a list of Rab proteins in mouse 430 2.0 and plotted based on CD8α⁺ DC/CDP (x axis) versus CD8α⁺ DC/CD8α⁻ DC (y axis) expression ratios. Each dot indicates an individual probe set. Data are representative of at least two independent experiments with three pooled mice. (B) Expression value (in arbitrary units) of Rab43 mRNA normalized to Hprt by quantitative RT-PCR for the indicated cell populations. Data from three independently sorted replicates of three WT mice displayed as mean ± SEM are shown. (C) Immgen data showing expression of Rab43 in the indicated populations from sLN. Data are displayed as mean ± SEM with three measurements per sample. (D) Western analysis of RAB43 and β-actin for the indicated spleen or BM populations from WT mice. Data are representative of at least three independent experiments. Mac, macrophage; Mono, monocyte. (E) Intracellular staining for RAB43 in the indicated cells from spleen and sLN from WT and Rab43^Δ/Δ B6 mice. The numbers represent the mean fluorescence intensity of RAB43 staining for the indicated cells. Data are representative of two independent experiments. (F) Western analysis for RAB43 and lamin B from WT or Rab43^Δ/Δ 129 (Δ/Δ) splenocytes. CD11c-negative (−) or CD11c-positive (+) splenocytes were isolated using CD11c microbeads. Data are representative of at least two experiments. (G) Western analysis for RAB43 and lamin B from CD11c-negative (−) or CD11c-positive (+) B6 splenocytes isolated as in A derived from Rab43^f/f mice that were either CD11c^Cre− (Cre-) or CD11c^Cre+ (Cre+) as indicated. Data are representative of at least two experiments. (D, F, and G) Scales indicate molecular weight in kD. (H) Percentage (left) and absolute number (right) of DC subpopulations from spleen of WT and Rab43^Δ/Δ B6 mice. (I) Percentage (left) and absolute number (right) of DC subpopulations from sLN of WT and Rab43^Δ/Δ mice. Cells gated based on resident (B220⁻MHCII^intCD11c^hi) and migratory (B220⁻MHCII^hiCD11c^int/lo) populations are shown. (H and I) Data from three independent experiments are shown. Each dot represents a single mouse. (J) Contour plots of tissue DCs from the small intestine lamina propria (SILP) or liver of WT or Rab43^Δ/Δ 129 mice pregated on B220⁻CD45.2⁺MHCII⁺CD11c⁺. Data are representative of at least two experiments. (K) Percentage of IL-12– and TNF-positive cells after incubation of FLT3L-cultured BM cells from WT and Rab43^Δ/Δ 129 mice with LPS, CpG, polyI:C, or STAg. Data from two independent experiments displayed as mean ± SEM are shown.