Purification of EAP. (A) Schematic flow chart of the purification procedure. (B) Composition of EAP. EAP was purified from approximately 5 × 10⁸ 293fENL cells. As a mock control, the same purification strategy was performed using unliganded agarose in place of flag-agarose for the first precipitation step (mock). Eluates were run on a 10% SDS-PAGE, stained with Coomassie (left panel) and subsequently with silver (right panel). The bands were excised, digested, and analyzed by mass spectrometry. All proteins that could be uniquely identified by more than 1 peptide and at least in 2 independent experiments are listed. Where individual band identities could be assigned, they are indicated in the figure. (C) Left panel shows nucleic acid content of cellular extracts used for EAP purification. Nucleic acids from cellular extracts used for EAP purification were isolated by phenol extraction, separated by agarose gel electrophoresis and stained by ethidium bromide. Samples shown are from untreated cellular extracts (extract) and from extracts after nuclease digestion (nuclease). Right panel shows purification of EAP from nuclease-treated cell extracts. EAP was precipitated as described in panel B from cellular extracts after exhaustive nuclease digestion. The figure shows a silver stained gel of EAP proteins in com-parison to a mock control as described in panel B. (D) Coimmunoprecipitation of major EAP components with endogenous ENL. Immunoprecipitations from HEK293 cell extracts were done either with agarose coupled to anti-ENL antibodies (α-ENL) or flag-agarose (control). Coprecipitating proteins were analyzed by immunoblot with antibodies against the pTEFb subunits CDK9, CYCT2, and HSP70, as well as with antibodies specific for DOT1L and AF4. The 2 splice variants of CYCT2 (CYCT2a, 2b) are indicated.