Ni2 nta agarose

Recombinant baculovirus carrying deletion mutants of USP9X was generated with the Bac-to-Bac System (Invitrogen). Infected Sf9 cells were grown in spinner culture for 48–96 h at 27 °C and His-tagged proteins were purified using Ni²⁺-NTA agarose (Invitrogen) according to the standard procedures. Full length or deletion mutants of CEP131 were purified from bacteria BL21 cells with Glutathione-agarose.

Recombinant baculovirus carrying full-length USP7/WT or deletion mutants of USP7 were generated with the Bac-to-Bac System (Invitrogen). Infected Sf9 cells were grown in spinner culture for 48–96 h at 27°C and His-tagged protein-purified using Ni²⁺-NTA agarose (Invitrogen) according to standard procedures. For the His pull-down assay, His-tagged protein was incubated with recombinant EZH2, SUZ12, or EED that was in vitro-transcribed and translated according to the manufacturer’s procedures (TNT T7 Quick Coupled Transcription/Translation Kit; Promega, Leiden, the Netherlands) at 4°C overnight. GST-fusion proteins were purified from Escherichia coli by glutathione-Sepharose 4B beads (GE Healthcare) and then washed with high salt buffer (20 mM Tris-HCl pH 7.4, 0.1 mM EDTA, and 300 mM NaCl). For the GST pull-down assay, GST-fusion proteins were incubated with in vitro transcribed and translated proteins at 4°C overnight. The beads were washed three times, then boiled in SDS loading buffer, and subjected to SDS-PAGE followed by immunoblotting.