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Fig. 6. Lysosomal targeting of Arf6-pathway cargo is enhanced in 
-adaptin-depleted cells. (A) Internal pools of MHCI were measured by uptake of Alexa-Fluor-488-conjugated anti-MHCI antibody for 2 hours at 37°C. Antibody remaining at the cell surface was quenched using anti-Alexa-Fluor-488 antibody, and the internal pool was quantified by flow cytometry. Levels in clathrin and -adaptin-depleted cells are reported relative to those in control cells. Data represent results from four independent experiments, each performed in duplicate; **P=0.0003, statistically significant difference. (B) Cell extracts were prepared from control or -adaptin-depleted cells, treated with bafilomycin A1 (BafA) at 250 nM or not treated, as indicated. Immunoblotting was performed using antibodies against MHCI, -adaptin and p70S6k. (C-E) Cell-surface proteins were biotinylated at 4°C, then returned to 37°C for 4 hours. Biotinylated proteins were immobilized using NeutraAvidin beads (Pdn), and subjected to immunoblotting with the indicated antibodies. In C, t=0 samples were immunoblotted with anti-TfnR antibody to confirm significant inactivation of clathrin and AP-2. In D and E, anti-MHCI immunoblotting was performed on cells depleted of -adaptin (D) or clathrin (E). WCL, whole-cell lysate. (F) Quantification of MHCI levels from biotinylation experiments. Levels remaining after the 4-hour chase period were normalized against the starting levels (t=0) for each respective siRNA. Data represent results from at least three independent experiments, each performed in duplicate.**P values (ranging from P=0.001 to P=0.01) indicating statistically significant differences.
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