Expression in Freestyle 293-F cells

For immunoprecipitation assays, plasmids containing centrobin constructs with a 5xMyc tag and TRIM37 constructs with a 3xFLAG tag were transfected into FreeStyle 293-F cells (Thermo Fisher) using FreeStyle MAX Reagent and OptiPRO SFM according to manufacturer guidelines (Thermo Fisher). Each transfected sample was a 20 ml culture (10⁶ cells/ml). Equal amounts (12.5 μg) of each expression plasmid were used for co-transfections. Following transfection, FreeStyle 293-F cells were incubated for 48 hours on an orbital shaker platform (125 rpm) at 37°C in 8% CO₂. 10 ml of each sample were collected and washed with Dulbecco’s phosphate buffered saline (DPBS; Thermo Fisher). Cells were collected by pelleting and resuspended in 1 ml of lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EGTA, 1 mM DTT, 2 mM MgCl and one EDTA-free protease inhibitor cocktail tablet (Roche). Cells were sonicated in a water bath sonicator at 4°C for 6 minutes to generate a crude lysate. The crude lysate was centrifuged at 13,000 rpm for 15 minutes at 4°C in a microfuge to generate supernatant and pellet fractions. Immunoprecipitations were performed by adding 20 μl of Pierce anti-Myc magnetic beads to 1 ml of supernatant (see Table S3). After incubating for 2 hours on a rotator at 4°C, beads were washed 5X in 900µl of lysis buffer and resuspended in 60 μl of 4x Laemmli sample buffer. For coimmunoprecipitation assays detecting ubiquitination, FreeStyle 293-F cells were transfected and incubated with equal amounts (8.5 μg) of three DNA constructs encoding: Myc-centrobin, FLAG-TRIM37, and haemagglutinin (HA)-tagged ubiquitin. For these IPs 5 mM N-ethylmaleimide was added to the lysis buffer.