Protein a g slurry

Five to ten micrograms of anti-ubiquitin, anti-PDK1, and anti-COL11A1 antibody were added to an Eppendorf tube containing 500 μg of cold, pre-cleared protein lysate and incubated at 4°C overnight, and then 35 μL of washed, Protein A/G slurry (Santa Cruz) in pre-chilled lysis buffer was added the next day. The supernatant was carefully and completely removed, and the beads were washed 3–5 times with 500 μL of lysis buffer. After the last wash, the supernatant was aspirated, 50 μL of 1 × sample buffer was added to the bead pellet, and the sample was boiled for 10 min. After spinning at 13,000 × g for 5 min, the supernatant was collected for 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)].