Journal: Traffic (Copenhagen, Denmark)
Article Title: Two Di-Leucine Motifs Regulate Trafficking of Mucolipin-1 to Lysosomes
doi: 10.1111/j.1600-0854.2006.00387.x
Figure Lengend Snippet: (A) HeLa cells were transfected with siRNA targeted to control (non-silencing) or to the µ2 subunit of the AP2 complex. Seventy-two hours after the second round of transfection, equivalent amounts of homogenate from control and siRNA-treated cells were subjected to SDS-PAGE and immunoblotted using antibodies to the β2 subunit of AP2 or annexin-II (loading control).(B) Control and siRNA-treated HeLa cells were incubated with rhodamine-transferrin for 15 min at 37 °C and analyzed by confocal microscopy. (C) HeLa cells treated with non-targeted siRNA or siRNA against AP2 were transfected with Tac-MLN-Ctail, and the amount of the chimera present at the plasma membrane was quantified by FACS analysis. Results are expressed as the percent of Tac-MLN-CTail remaining at the cell surface after 15 min incubation at 37 °C. Values represent the mean ± SD from two independent experiments.(D) HeLa cells were co-transfected with Tac-MLN-CTail and AP2-GFP, incubated for 1 h on ice with monoclonal antibody to the extracellular domain of Tac, washed and allowed to internalize for 1 min at 37 °C. Cells were then fixed, permeabilized, stained with a Cy3-conjugated donkey anti-mouse IgG and analyzed by confocal fluorescence microscopy. Insets show a fourfold magnification of the indicated region. Bars represent 10 µm.
Article Snippet: The following antibodies were used: mouse monoclonal anti-EEA1 (BD Transduction Laboratories, San Jose, CA, USA), mouse monoclonal anti-CD63 (H5C6; Pharmingen, San Jose, CA, USA), mouse monoclonal anti-β subunit of AP2 (100/2; Sigma-Aldrich, St Louis, MO, USA), rabbit anti-GFP (MBL International, Woburn, MA, USA), mouse monoclonal anti-GFP (ab1218; Abcam, Cambridge, MA, USA), mouse monoclonal anti-IL2 Receptor alpha (Tac) (143–13; Abcam) and mouse monoclonal anti-annexin-II (Zymed, San Francisco, CA, USA).
Techniques: Transfection, SDS Page, Incubation, Confocal Microscopy, Staining, Fluorescence, Microscopy
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