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. 2018 Jan 29;4:4. doi: 10.1038/s41420-017-0003-8

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Identification of the MID1-interactome. MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation. MID1-binding proteins were identified by mass spectrometry. The mTOR-dependent translation initiation pathway is shown and the number of proteins identified belonging either to the eukaryotic translation initiation factor complex (eIF complex) or the ribosome are indicated. b Validation of the mass spectrometry results shown in a and Table 1. MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation (IP FLAG). As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies to detect MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. c Effect of ribosome disassembly on the composition of the MID1-complex. MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of high concentrations of EDTA. Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, RPLP0, RPL5, RPS3. d To analyze the MID1-complex composition and its dependency on RNA, MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of RNAse. As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. e, f mTOR regulates translation of APP. e In vitro translation of in vitro transcribed APP-mRNA tagged to luciferase in the presence or absence of the mTOR-inhibitor temsirolimus. The level of translated luciferase reporter was measured in a luciferase assay. Columns represent mean values ± SEM. n = 3. *p < 0.01. f Primary neurons were treated with the mTOR-inhibitor temsirolimus. Protein extracts were analyzed on western blots, detecting APP and β-actin as loading control. Graph shows quantification of western blots, mean values ± SEM. n = 3. *p < 0.01. APP amyloid precursor protein