293T cells overexpressing either C7orf26_V1 or C7orf26_V2 were prepared using lentivirus and selected using puromycin as described above. Parental cell lines with no overexpression construct were passaged in parallel for the ‘Mock’ control. For large scale purifications, 5 plates of confluent 15 cm plates were harvested and subjected to the nuclear lysate protocol described above. The resulting lysates for each condition were diluted to 1 mg / mL in EB300.
For HA purifications, 5 mL of nuclear lysate from each condition was incubated with 250 uL of Pierce HA Magnetic Beads (Thermo Fisher, 88836) and rotated overnight at 4C. Afterwards, beads were collected using a magnetic rack and washed 6× with EB300 and 2× with PBS buffer all at 4C. We eluted precipitated protein from the beads using low pH elution in glycine buffer as described (https://www.abcam.com/protocols/immunoprecipitation-protocol-1#elution ). For V5 purifications, the same protocol was performed but with Anti-V5 Agarose Affinity Gel (Sigma-Aldrich, A7345–1ML). A portion of samples were saved for immunoblot analysis, and the remainder was to mass spectrometry with the Taplin Mass Spectrometry Facility, the full details of which are previously described under “Sample Preparation” (Mashtalir et al., 2018 (link)). The resulting total peptide counts for each protein were reported.
For HA purifications, 5 mL of nuclear lysate from each condition was incubated with 250 uL of Pierce HA Magnetic Beads (Thermo Fisher, 88836) and rotated overnight at 4C. Afterwards, beads were collected using a magnetic rack and washed 6× with EB300 and 2× with PBS buffer all at 4C. We eluted precipitated protein from the beads using low pH elution in glycine buffer as described (