Fig 1: Rab7b depletion does not affect DC maturation and antigen presentation ability. (A) MDDCs were transfected by electroporation with either control siRNA or Rab7b siRNA. 18 h (for immature DCs; imDCs) or 48 h (for LPS-DCs) after transfection the DCs were harvested, lysed and subjected to western blot analysis with antibodies against Rab7b and tubulin as a loading control. (B) Quantification of Rab7b levels in MDDCs silenced with control siRNA or Rab7b siRNA. The intensity of the bands from western blots was quantified using ImageQuant, and the level of Rab7b was normalized to the amount of tubulin. Data represent the mean±s.d. of three independent experiments. ***P<0.0001 (two-tailed unpaired Student's t-test). (C) FACS analysis of the surface expression markers CD80, CD86 CD83 and CD11c (left panels), and HLA-DR, HLA class I and CCR7 (right panels). A representative histogram overlay is shown for each marker. The black line represents mock electroporated (without siRNA) MDDCs, the blue line the control siRNA and the red line the Rab7b siRNA electroporated MDDCs. The gray line corresponds to isotype antibodies. The x-axis represents the mean fluorescence intensity of the conjugated markers indicated for each histogram. The histograms are representative examples from one out of three independent experiments. All experiments were repeated three independent times. (D) Radium-1 TCR-expressing T cells were stimulated for 5 h with either control siRNA- or Rab7b siRNA-treated DCs loaded with a specific 19-mer peptide encoded by the TGFBR2 frameshift mutation. An anti-CD107a antibody was used to assess the amount of degranulation by CD8+ cytotoxic T cells specifically activated by DCs. Data represents the mean±s.d. of three independent experiments.