Pfn21a vector

Full-length AR and AR truncation mutants were cloned into pFN21A vectors (Promega) in accordance to the manufacturer’s instructions. After cloning, the fusion proteins were expressed using the TNT® SP6 High-Yield Wheat Germ Reaction cell-free transcription and translation system (Cat.# L5030, Promega) following the manufacturer’s protocol. For each reaction, protein expression was confirmed by immunoblotting. Purified menin protein was kindly provided by Dr. Tomek Cierpicki. Purified AR was purchased from Prolias Technologies (R1089-2), purified ASH2L was purchased from Cayman Chemicals (Cat. No. 10946), Purified Max and Ring1B protein was kindly provided by Dr. Xiaoju Wang.
For in-vitro purified pull-down experiments, 100ng AR was incubated with 20ng purified protein in binding buffer (20mM Tris-Cl (pH 7.5), 150mM NaCl, 0.1% Triton X-100 (vol/vol), 1 mM dithiothreitol and protease inhibitors) and incubated at 4°C for 1 hr. AR was then immunoprecipitated and bound proteins were analyzed by western blotting as described above.
For interaction between menin and AR truncation mutants, 200–250ng purified menin was incubated with Halo-tagged FL-AR and truncation mutants in binding buffer. Menin was immunoprecipitated and bound proteins were analyzed by immunoblotting with anti-Halo antibody.

EZH2, BMI1, RING1B, EED and sub-domains were cloned into pFN19A and pFN21A vectors (Promega) in accordance to the manufacturer’s instructions. Fusion proteins were expressed in TNT® SP6 High-Yield Wheat Germ Reaction (Promega) following manufacturer’s instruction, or transfected into HEK293 cells for transient expression. A total of 2.0μl of cell-free reaction containing the HaloTag® fusion protein or 10ug total proteins from HEK293 cell lysates were resolved on SDS gels and blotted using anti-Halo or indicated antibodies (Supplementary Table 3) Dilutions are listed in Supplementary Table 3.