Flag ha modified pcdna3.1 vector

Phf5a ORF was cloned into the pGEX-6P-1 vector (GE Healthcare) (kind gift from Dr. K.J. Armache, NYU School of Medicine) and transformed into BL21 Star (DE3) bacteria. 12L of liquid cultures were induced overnight with 0.1mM IPTG at 18°C. Bacteria were lysed in 20mM Tris-HCl pH8.0, 200mM NaCl, 1mM DTT passing though a pressure homogenizer. Soluble fraction was bound to glutathione agarose beads (Pierce) for 1h at 4°C, beads were washed with 200 column volumes (CV) Lysis Buffer and GST-Phf5a protein was eluted using reduced glutathione. GST tag was cleaved by cleavage with PreScission Protease (kind gift from Dr. K.J. Armache, NYU School of Medicine) and Phf5a was further purified by ion exchange and size exclusion FPLC chromatography. Paf1, Cdc73 and Wdr61 were cloned in Flag/HA-modified pCDNA3.1 vector (Invitrogen) and in vitro translated using TNT Coupled Wheat Germ Extract System (L4140, Promega). 5µg of purified Phf5a protein was added, mixtures were bound overnight at 4°C and HA-tagged proteins were pulled-down using HA affinity gel beads (Invitrogen) for 4h at 4°C. Beads were washed 4 times with 1mL Lysis Buffer and interactions were visualized by WB analysis.