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. Author manuscript; available in PMC: 2013 Mar 13.
Published in final edited form as: Mol Cell. 2012 Sep 27;48(3):445–458. doi: 10.1016/j.molcel.2012.08.022

Figure 2. PBAF, LSD1, PHF8, and AF4p12 Associate with ICN1-CSL-MAML1.

Figure 2

(A) FLAG and HA-immunopurified ICN1-associtated proteins from SupT1 nuclear extracts (NEs) were resolved on SDS-PAGE and the presence of partners identified by mass spectrometry was confirmed by Western blot (WB).

(B) NEs from SupT1 stably expressing LSD1-F (FLAG-tagged), PHF8-F or BRG1-F were subjected to immunoprecipitation (IP) using anti-FLAG beads. The presence of endogenous ICN1 and CSL in the purified material was revealed by WB. The anti-ICN1 antibody specifically recognizes the γ-secretase-cleaved active form of NOTCH1 (V1744).

(C) Interaction between endogenous LSD1, PHF8 or BRG1 with components of the Notch-activation complex. LSD1, PHF8, and BRG1 were purified from SupT1 NEs using specific antibodies and the presence of ICN1 or CSL in the purified material was revealed by WB.

(D) SupT1 cells stably expressing FLAG and HA-tagged MAML1 (MAML1-F/H) were treated with DMSO or GSI (500 nM, 8 hr). MAML1-associated proteins were FLAG-HA immunopurified from NEs and analyzed by WB using the indicated antibodies.

(E) BRG1, PB1, LSD1, PHF8, and AF4p12 are associated with the Notch-activation complex. NEs from SupT1 stably transduced with FLAG-MAML1 and HA-ICN1 were subjected to sequential IP using anti-FLAG and anti-HA beads. The presence of Notch cofactors in the purified material was analyzed by WB.

(F) Notch cofactors assemble into a single complex containing ICN1-CSL-MAML1. Reciprocal IPs with anti-FLAG and anti-HA beads were performed using NEs from SupT1 cells stably coexpressing HA-ICN1 and FLAG-PHF8, -LSD1 or -BRG1. Eluates were subjected to WB.