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Sortilin and Spadin interact with the TREK-1 channel. Co-immunoprecipitation: Immunoprecipitation of Sortilin with anti-TREK-1 antibody (IP α-TREK-1) or immunoprecipitation of TREK-1 with anti- Sortilin antibody (IP α-Sort) from transfected COS-7 cells or mouse cortical neurons. Immunoprecipitated proteins are subjected to Western blots and revealed using anti-sortilin (WB: α-Sort) or anti-TREK-1 (WB: α-TREK-1). Addressing: Influence of Sortilin on the expression of TREK-1 at the plasma membranes. COS-7 cells are transfected with TREK-1 in the absence or in the presence of Sortilin. Crude homogenates, purified plasma membrane proteins or cell surface biotinylated proteins are subjected to Western blot analysis and revealed using anti-TREK-1 antibody. Binding: Competition between 125I–Spadin and unlabeled Spadin (white circles) or NT (black circles) for binding to TREK-1 transfected COS-7 cell homogenates. Internalization, Association kinetics of 125I-Spadin binding to COS-7 cells tranfected with TREK-1. At the indicated times, cells are either washed twice with 500 μL of binding buffer (black circles) or treated with 500 μL of acid-NaCl buffer for 2 min (white circles).
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