Fig. 5.

Prep1 reexpression affects Meis1a interacting proteins. (A) Purification of full-length Meis1a and Prep1 by SDS-PAGE. GST constructs were coupled to glutathion-sepharose beads and run on 8% (vol/vol) SDS-PAGE. Asterisks show the migration of GST and each GST construct in Coomassie blue-stained gel. (B) Pull-down analysis of purified Meis1a and Prep1 by GST and GST-Pbx1b by immunoblotting and Ponceau staining (indicated). (C) TAP–MS analysis of the Meis1a interactome. To define the Meis1a interactome in the absence and presence of Prep1, TAP-Meis1a was expressed in Prep1^i/i and Prep1^i/i MEFs reexpressing Prep1 and purified using glutathione-sepharose beads. Then, the purified complexes were eluted by tobacco etch virus (TEV) protease cleavage. The remaining complex after TEV cleavage was further purified using calmodulin beads and eluted by boiling in SDS sample buffer. The final TAP eluate was separated on a 10% (vol/vol) SDS-PAGE and stained by colloidal Coomassie blue. The entire lane was cut out from the gel and divided into different zones. Proteins identified by liquid chromatography–MS-MS complex compositions from each zone are listed. Exponentially modified protein abundance index (emPAI) is indicated as a measure of relative quantitation of proteins in each sample. A purification from cells infected with TAP empty vector is presented as control. (D) Ddx proteins coimmunoprecipitate with Meis1a; 300 μg nuclear extracts of the Prep1^i/i MEFs infected with either FLAG-Meis1a alone or FLAG-Meis1a and Prep1 was immunoprecipitated with M2 anti-FLAG affinity resins and immunoblotted with the anti-FLAG, anti-Ddx3x, and anti-Ddx5 specific antibodies. One-tenth of the lysates used for immunoprecipitation was loaded as input. (E) Ddx proteins do not coimmunoprecipitate with Prep1. FLAG-Prep1 was immunoprecipitated from 300 μg nuclear extracts of the Prep1^i/i cells infected with FLAG-Prep1 using M2 anti-FLAG affinity resins. The Prep1, Ddx3x, Ddx5, Meis1a, and Pbx1 in the inputs and precipitated samples were identified by immunoblotting using appropriate antibodies. IP, immunoprecipitation. (F) Pbx down-regulation decreases the Meis1–Ddx interaction. FLAG-Meis1a was immunoprecipitated from 300 μg nuclear lysate of Prep1^i/i cells overexpressing Meis1a-shCTRL or Meis1a-shPbx1 and immunoblotted with the anti-FLAG–, anti-Ddx3x–, anti-Ddx5–, and anti-Pbx1–specific antibodies; one-tenth of the lysates was loaded as input.