Functional dissection of the interactions of stonin 2 with the adaptor complex AP-2 and synaptotagmin -- Data Supplement - HTML Page - 07862SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Materials and Methods
Antibodies.
Mouse monoclonal antibodies against clathrin light chains, synaptotagmin I (Cl41.1), and synaptophysin were a kind gift of R. Jahn (Max Plank Institute for Biophysical Chemistry, Göttingen, Germany), antibodies against clathrin heavy chain, dynamin, auxillin, and AP180 were kindly provided by P. de Camilli (Yale University, New Haven, CT). Antibodies against a -adaptin, b 1/b 2-adaptin, m 2-adaptin, m 3-adaptin, and hsc70 were obtained from Transduction Laboratories (Lexington, KY) or Affinity BioReagents, (Golden, CO). Antibodies against m 1-adaptin were a generous gift of P. Schu (University of Göttingen). a HA-antibodies were obtained from Affinity BioReagents, Sigma-Aldrich, and Berkeley Antibody. Peptides.
HPLC-purified WVxF-containing peptide (WVTFDDD) or a mutant thereof (AVTADDD) were purchased from Coring System Diagnostics (Heidelberg). Immunoprecipitation.
For immunoprecipitations from rat brain, cytosolic or synaptosomal membrane fractions were solubilized in 10 mM Hepes-KOH (pH 7.4)/100 mM NaCl/1% Triton X-100/1 mM PMSF, and were precleared by ultracentrifugation. Samples were incubated with monoclonal antibodies prebound to protein G-Sepharose for 4 h at 4°C, were washed extensively, and were analyzed by SDS/PAGE and immunoblotting. For immunoprecipitation from transfected COS7 cells, cells were harvested from a 10-cm dish 24 h posttransfection, and were solubilized in lysis buffer (20 mM Tris•HCl, pH 7.5/150 mM NaCl/5 mM EDTA/1% Triton X-100/1 mM PMSF/1 mM DTT). After centrifugation at 13,000 ´ g, supernatants were incubated with a HA-antibody (Babco, Richmond, CA) coupled to protein G agarose for 4 h at 4°C. Following extensive washing, bound proteins were eluted and analyzed by immunoblotting. For immunoprecipitations from multiple tissue extracts (100 m g of total protein), lysates were incubated with stonin 2 antisera prebound to protein G-Sepharose beads in a buffer consisting of 1% Triton X-100, 0.2% SDS, 20 mM Hepes, 100 mM NaCl, 2 mM MgCl₂, and 1 mM PMSF. Eluted protein was separated by SDS/PAGE and was analyzed by immunoblotting.