Flag-HA fusion construct, microinjection, immunoprecipitation, mass spectrometry

Fusion construct and microinjection performed as described above. Positive injection was confirmed by Dot Blot (Furrer et al, 2017; Rzeszutek et al, 2022). Immunoprecipitation was performed as described before (Reuter et al, 2009; Hoehener et al, 2018). Non-crosslinking was performed because the IP of SMC4-2 under crosslinking did not work at pH below 10.4. In detail, 400-ml cells were harvest at 4 h after 100% fragmentation, pellets were resuspended in 2 ml fresh lysis buffer (50 mM Tris pH 8.8, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 1% Triton X-100, 1× protease inhibitor complete tablet [Roche], and 10% glycerol) and sonicated until complete lysis. The cell lysates were spin down at 13,000g, 4°C for 30 min 1 ml of the supernatant was incubated with 50 μl of Anti-HA affinity resin (Roche) overnight at 4°C while rotating. Another 1 ml supernatant was frozen in liquid nitrogen and store at −80°C for later using. Beads were washed with 1 ml IP buffer (10 mM Tris pH 8.8, 150 mM NaCl, 0.01% NP-40, 1 mM MgCl₂, 1× protease inhibitor, and 5% glycerol) for three times before incubation. After overnight incubation, beads were washed with 1 ml IP buffer for five times. Washed beads were resuspended in 50 μl IP buffer, boiled with 25 μl 5× SDS loading buffer at 95°C, after cooling down on ice, immediately used for Western blot and mass spectrometry analysis in the University of Bern.