Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - EPSIN1 is not permanently associated with clathrin coated vesicles. Leaf tissues were homogenized in grinding buffer (40 mM Hepes-KOH, pH 7.5, 10 mM KCl, 3 mM MgCl₂, 0.4 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 0.02% NaN₃) supplemented with EDTA free protease inhibitor cocktail. Homogenates were centrifuged at 14,000xg for 5 min to remove debris, and the supernatant was separated into soluble and membrane fractions by ultracentrifugation at 100,000xg for 1 h. The pellet fraction was resuspended in the homogenization buffer supplemented with 1.0% Triton X-100 and applied to a Sephacryl S-300 high-resolution column. Proteins were eluted using buffer (50 mM sodium phosphate, pH 7.0, 0.15 M NaCl, 0.02% NaN₃) at a flow rate of 0.5 ml/min in a fraction volume of 3.0 ml. Fractions were analyzed by Western blotting using an anti-clathrin, anti-VSR and anti-EPSIN1 antibodies. The size of proteins in the eluted fractions was determined using thyroglobulin (669 kD), apoferritin (443 kD) and BSA (66 kD).