Figure 4 (IMAGE)
Caption
LC-MS/MS analysis identifies putative MNK1/2 targets and interactors. (A) FLAG-MNK2 or FLAG-MNK1 was overexpressed in 293T cells. MNK1/2 was immunoprecipitated from 293T cell lysates using anti-FLAG-M2 agarose conjugated beads. An empty vector was used as a negative control. Immunoprecipitated proteins were resolved by SDS-PAGE, and were prepared using standard techniques and then analyzed via LC-MS/MS. A Venn diagram was created depicting the number of proteins that interact with MNK1/2. (B) The results from (A) were annotated using Metascape. The heat map shows the most significant pathways and the overlap between the MNK1 IP and MNK2 IP. (C) FLAG-MNK2 or FLAG-MNK1 was immunoprecipitated as described in (A). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) FLAG-MNK2 was overexpressed in 293T cells. Cells were treated with either DMSO (vehicle-control) or Tomivosertib at the indicated doses and time points, lysed and MNK2 was immunoprecipitated with anti-FLAG-M2 agarose conjugated beads. An empty vector (EV) was used as a negative control. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (E) HA-RAPTOR was overexpressed in 293T cells. Cells were treated with either DMSO (vehicle-control) or Tomivosertib at the indicated doses and time points, lysed and RAPTOR was immunoprecipitated with anti-HA Sepharose conjugated beads. An empty vector (EV) was used as negative control. Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (C-E) Total cell lysates for each experimental condition were run in parallel with the immunoprecipitated proteins (Input).
Credit
Correspondence to - Leonidas C. Platanias - l-platanias@northwestern.edu
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Copyright: © 2021 Suarez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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