Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation
Figure 3
Cargo Overexpression in a Tetracycline (tet)-Repressed System
BSC1 cells, coinfected with adenoviruses coding for a tet-repressible transcription activator and the human transferrin receptor (TfnR), were cultured in the presence (+) or absence (−) of tet.
(A) Validation of infection efficiency by adenoviruses: cells were loaded with Tfn-Alexa 568, and analyzed by epifluorescence. The scale bars represent 10 μm.
(B) Whole-cell lysates were analyzed by SDS-PAGE and western blot.
(C) Quantitative analysis of surface-bound TfnRs in BSC1 cells stably expressing LCa-EGFP and σ2-EGFP under control (+ tet) or cargo overexpressing (− tet) conditions. Error bars represent standard deviations of three independent experiments.
(D) CCPs, visualized by LCa-EGFP and TIR-FM, in control (ctrl) and TfnR overexpressing (cargo o/x) cells. The scale bar represents 5 μm.
(E) Average pit density in LCa-EGFP– and σ2-EGFP–expressing cells under control and cargo overexpressing conditions.