Immunoprecipitation Workflow Optimization

For immunoprecipitation experiments, cells were lysed in 140 mM NaCl, 10 mM Tris pH 7.6, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, protease and phosphatase inhibitors. Cells were incubated on ice for 30′, then centrifuged for 20′ at 4 °C, at 13000 rpm. Thereafter, supernatant (1–2 mg) underwent immunoprecipitation as described^{33 (link)}, by using 4 μg of each antibody or control IgGs (mouse IgGs were from Santa Cruz, cat. #sc-2025; rabbit IgGs were from Merck, #cat. I8140). To avoid IgGs detection, either ImmunoCruz IP/WB Optima B, C, F, E (Santa Cruz Biotechnology; cat. #sc-45039, cat. #sc-45040, cat. #sc-45043, cat. #sc-45042, respectively) or the VeriBlot reagent (Abcam; #cat. ab131366 and #ab131368) were used in western blot, after the immunoprecipitation procedure, according to the manufacturer’s instructions.