Supplemental Data Ligands for Clathrin-Mediated Endocytosis Are Differentially Sorted into Distinct Populations of Early Endosomes

Immunofluorescence Cells were fixed in 2% (v/v) formaldehyde for 40 min. After being washed in PBS, the cells were permeabilized in a blocking buffer containing 10% (v/v) FBS, 3% (w/v) BSA and 0.5% (v/v) Triton X-100 in PBS. The cells were then incubated at 4 °C with primary antibodies against EEA1, CI-MPR, clathrin heavy chain or the α subunit of AP-2 for 6-12 hours. Excess antibody was removed by extensive washing with PBS containing 0.2% (w/v) BSA and 0.1% (v/v) Triton X-100, and the cells were then incubated at room temperature for 30 min with secondary antibodies (Molecular Probes). For immunofluorescence experiments involving transferrin, experimental procedures are similar except that the blocking buffer used contains 0.1% Tween 20 and 5% BSA and the wash buffer contains 0.1% Tween 20 and 1% BSA. For immunofluorescence against Dab2, cells were extracted with 0.03% saponin in cytosolic buffer (25mM Hepes, pH 7.4, 25 mM KCl, 2.5 mM magnesium acetate, 5 mM EGTA and 150 mM Kglutamate) for 1 min prior to fixation with 4% paraformaldhyde for 20 min. These cells were then permeabilized in 0.1% Triton X-100 for 10 min, blocked in PBS containing 1%BSA for 60 min, and then incubated with primary antibodies for 90 min and the appropriate secondary antibodies for 60 min.