Cell Lysis and Immunoblotting Protocol

Cells were lysed in 20 mM HEPES (pH 7.5) buffer containing 0.5% Nonidet P-40 (NP-40), 50 mM KCl, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, and protease inhibitors. Soluble lysates were subjected to SDS-PAGE, and then immunoblotted with the appropriate antibodies. Secreted proteins from cell culture supernatants were concentrated for immunoblotting as described previously.^{17 (link)} All the blots were the representative image of at least three-independent experiments. For the coimmunoprecipitation experiment, cells were lysed in 10 mM HEPES buffer (pH 7.4) containing 0.2% NP-40, 100 mM KCl, 5 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT, and protease inhibitors. Lysates were pre-cleared with Protein G sepharose 4 beads (GE) and immunoprecipitated with the indicated antibodies, and the bead-bound proteins were immunoblotted with the appropriate antibodies.