Mouse anti flag m2 antibody conjugated beads

Immunoprecipitation were performed as previously described (25 (link)–27 (link)). Briefly, for transient transfections, HEK293T cells were plated at a density of 5–7 × 10⁵ cells/well in six-well plates and cultured for 24 h. The cells were transfected with DNA constructs using calcium phosphate (Clontech laboratories, Mountain View, CA, USA) and incubated for 24 h. For FLAG immunoprecipitation, the transfected HEK293T cells were washed twice with PBS and lysed with a buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), and a protease inhibitor cocktail (Roche, Basel, Basel-Stadt, Switzerland). The cell lysates were incubated with 50 μl (bead volume) of mouse anti-FLAG M2 antibody-conjugated beads (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. The beads were subsequently washed three times with lysis buffer. The immunoprecipitates were eluted by adding 2 μg/ml of 3× FLAG peptides and analyzed by Coomassie blue staining.