Hot-wired endocytosis can function independently of AP2. (A) Chemically induced endocytosis can operate independently of endogenous AP2. Live-cell confocal immunolabeling experiments of cells expressing CD8–dCherry–FRB (dark mCherry variant) with FKBP–β2–GFP. Cells were also transfected with siRNAs against GL2 (control) or μ2 subunit of AP2 complex. Antibody feeding (internalized anti-CD8/Alexa 568) is shown to assess internalization. Inhibition of uptake of transferrin–Alexa 647 was used as a functional test of knockdown efficacy. Bar, 10 µm. (B) Quantification of chemically induced endocytosis. Fraction of above-threshold puncta that were green (FKBP–β2–GFP) and red (A568-CD8) is shown for each cell (spots); bars indicate mean ± SD. Number of experiments = 3. ANOVA, P = 1.48 × 10−8. Tukey test for siGL2 versus siμ2, P = 0.5. (C) Western blot (WB) to assess depletion of μ2 by RNAi. Cells were prepared in parallel with the experiment shown in C. Blotting for μ2 or for GAPDH (as a loading control) is shown.
