Figure 3.

Rab43^Δ/ΔCD8α⁺ DCs have normal Golgi development. (A and B) Sorted Ly6G⁻Ly6C⁻Thy1.2⁻B220⁻DX5⁻F4/80⁻CD11c⁺ splenocytes from WT (A) or Rab43^Δ/Δ (Δ/Δ; B) 129 mice were allowed to attach to coverslips, fixed, and stained for CD8α (white), giantin (red), and RAB43 (green). Coverslips were attached to slides using ProLong Gold antifade with DAPI (blue). Data are representative of at least three independent experiments. Bars, 5 µm. (C) Area of Golgi staining from WT and Rab43^Δ/Δ DCs. Each dot represents a single cell from staining in A and B, with 15 cells analyzed per genotype. Area was obtained using ImageJ. Statistics were analyzed by a two-tailed Mann-Whitney U test with P > 0.5. (D and E) Electron microscopy of sorted B220⁻CD11c⁺MHCII⁺CD24⁺CD172a⁻ splenocytes from Rab43^f/f-CD11c^Cre− (D) or Rab43^f/f-CD11c^Cre+ (E) B6 mice showing normal Golgi development. Data are from analysis of cells from five pooled mice. Bars, 500 nm. (F) Western analysis for LAMP1 in splenocyte protein lysate given no treatment (No Tx), EndoH, or PNGaseF. Data are representative of three independent experiments. The scale indicates molecular weight in kD. (G and H) Sorted SiglecH⁻CD11b⁻Sirpa⁻Bst2⁻ cells from FLT3 cultures of WT (G) or Rab43^Δ/Δ (H) B6 BM were incubated with Alexa Fluor 647–labeled HKLM-OVA (white) and then prepared as in B without CD8α stain (Videos 1 and 2). The yellow arrowhead indicates a RAB43-deficient DC that has taken up HKLM-OVA. Images are representative of three experiments. Bars, 5 µm. (I) Immgen expression data for cathepsin D in red pulp macrophages (RP MF), CD8α⁺ DCs, and CD8α⁻ DCs, displayed as mean ± SEM with three to four measurements per cell type. (J) Western analysis for cathepsin D (top) and β-actin (bottom) in BM macrophages (BMM), CD8α⁺ DCs, and CD8α⁻ DCs from Flt3L or M-CSF cultures either treated or untreated with LPS for 24 h. The scale indicates molecular weight in kD.