Immunoprecipitation and Western Blot Analysis

Cells were lysed in a buffer containing 50 mM Tris-HCl-pH8.0, 150 mM NaCl, 1 % NP-40, 1 mM Na₃VO₄, 1 mM PMSF, and protease and phosphatase inhibitors for 2 h at 4 °C. Lysates were centrifuged (45 min, at 45000 rpm, at 4 °C) and stored at −20 °C. Protein content was determined using Micro Bicinchoninic acid assay. For immunoprecipitation (IP), cell lysates (500 μg of total protein) were pre-cleared using 25 μl protein A/G slurry for 30 min at RT. The supernatants were then incubated with 3 μg mSos1, Cbl or isotype matched IgG Abs for 1 h at RT. 40 μl of protein A/G slurry (Santa Cruz Biotechnology, Santa Cruz, CA) were then added to the supernatants and incubated at 4 °C overnight. The bound proteins were resolved on 5–20 % SDS-PAGE. Western blot (WB) was performed using primary Abs and appropriate secondary Abs [26 (link)]. TrueBlot® ULTRA secondary Ab (Rockland Immunochemicals, Limerick, PA) was used for Rac1 to avoid background from the light chain band. For detection of Eps8 ubiquitination by WB, 5 mM N-ethylmaleimide was added in the IP buffer to prevent the cleavage of polyubiquitin chains [9 (link)].