Journal: Traffic (Copenhagen, Denmark)

Article Title: A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

doi: 10.1111/j.1600-0854.2012.01354.x

Figure Lengend Snippet: Figure 1: Myosin Vb tail mutant affects the recycling pathway. M10+22E cells transiently expressing Myosin Vb-GFP alone (A) or together with Rab11-FIP2-mCh (D), M10+YFP cells transiently expressing Myosin Vb tail-CFP (B) and Rab11-FIP2-mCh (C) were fixed with 4% PFA and immunostained for langerin (A and D, DCGM4), Rab11A (A), early endosome (B and C, EEA1). Single labelings (first three images from left to right) and merged images (last right images) are shown. Scale bars represent 5 μm. Quantification of langerin-YFP fluorescence amounts were performed on Average Intensity Projection of 3D stacks using Imag J tools on control cells (E and F, blue column, n = 14 and 16, respectively) and Myo Vb tail-CFP or Rab11-FIP2-mCh expressing cells (E and F, red column, n = 15 each). YFP fluorescence relative intensities are presented as mean + SD. Immunoblotting (G) was used to confirm the levels of langerin (Lang), in M1022E cells (0), overexpressing MyoVb-Tail mutant (MVb), Rab11-FIP2-mCh (FIP2) or cells infected with Rab11Q70L adenoviruses. This was completed by experiments in the presence (CQ, right panel) or absence (0, right panel) of 50 μM chloroquine, as described in section Material and Methods. Proteins were separated by SDS-PAGE in 12% acrylamide gels and blotted onto membranes, with equivalent amounts of protein being loaded in each lane. The membranes were also probed for β-tubulin (βTub), as a control. Blots are representative of three independent experiments.

Article Snippet: The following antibodies were used as secondary antibodies: Cy3-coupled anti-mouse IgG for TfR and 6C4; Cy3-coupled anti-rabbit IgG for Rab11, Cy3-coupled antigoat for EEA1 (Santa Cruz).

Techniques: Mutagenesis, Expressing, Control, Western Blot, Infection, SDS Page