Figure 6.
The distribution of AP2, clathrin, and AP1 between soluble and insoluble pools in cells overexpressing chimeric receptors with tyrosine- or di-leucine-based internalization signals. HeLa cells transiently transfected with plasmids encoding Tac, TTMb, or TTγt3-t2 were subjected to positive selection as described in MATERIALS AND METHODS. (A) A fraction of the cells (106 in this representative experiment) was removed and stained with fluorescein-conjugated anti-Tac and then analyzed by flow cytometry. Fluorescence intensity profiles from the three positively selected pools of cells (thick lines) and from mock-transfected cells (dotted lines) are shown. (B) The remaining cells were lysed and fractionated into Triton X-100-soluble (S) and insoluble (P) pools, and equal aliquots (derived from 106 cells/lane) were subjected to SDS-PAGE and immunoblotted with antibodies to AP2, clathrin heavy chain, and AP1 as described in MATERIALS AND METHODS. The migration of each band, as well as the heavy chain from the anti-Tac antibody used for the cell isolation, were visualized using radioiodinated antibody and are indicated on the figure. TTM, TTMb; TTγ, TTγt3-t2.