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. 2020 Jun 5;11:2835. doi: 10.1038/s41467-020-16696-6

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© The Author(s) 2020

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. 1 — a BioID screen for Rab35 interacting proteins. Enrichment (BirA*-Rab35/BirA* LFQ ratio) of MTMR protein-derived peptides from two independent experiments (mean ± SD). b, c Pulldown from lysates of HEK293T cells overexpressing eGFP (ctrl) or the indicated eGFP-tagged MTMRs with immobilized GST-Rab35•GTPγS (T) or GDP (D). b Representative immunoblots for eGFP or FLAG (MTMR13). Input: 5 % (MTM1, MTMR1, MTMR2, MTMR5), 6 % (ctrl), 3 % (MTMR13) of total protein added. c Quantification of representative data in b. Bound protein/input is shown. Two-tailed paired Student’s t-test of proteins bound to Rab35•GTP vs. GDP (n = independent experiments): MTM1: n = 4, p = 0.0892, t = 2.48, df = 3; MTMR1: n = 5, p = 0.2314, t = 1.41, df = 4; MTMR2: n = 7, p = 0.0712, t = 2.189, df = 6; MTMR5: n = 4, *p = 0.0191, t = 4.618, df = 3; MTMR13: n = 10, **p = 0.0048, t = 3.712, df = 9. d Affinity capture of eGFP-Rab35 GTP (CA, constitutively active) or GDP (DN, dominant-negative/inactive) locked mutants from lysates of FLAG-MTMR13-overexpressing HEK293T cells. Immunoblot analysis for eGFP, FLAG, or β-actin of input (I) and bound (B) fractions. Ratio of FLAG-MTMR13 bound to CA/DN is 11.6. IP immunoprecipitation. Input (I): 3.5% of total protein. e Same as in d but with MTMR13-ΔDENN. d, e are representative of two independent experiments. f Top, MTMR13 domain organization. Bottom, affinity capture of endogenous eGFP-Rab35 from KI HeLa cells expressing full-length MTMR13 or the indicated MTMR13-domains analyzed by immunoblotting as in d. Input (I): 4% (PH-GRAM, PTP), 3% (DENN, PTP + CC), 1.5% (Full length, PH) of total protein. Representative of two independent experiments. g–i Pulldown from lysates of HEK293T cells co-expressing eGFP-MTM1, -MTMR1, or -MTMR2, and FLAG-MTMR13 or FLAG using immobilized active GST-Rab35•GTP. g Representative immunoblot as in d. Input: 5% of total protein. h, i Quantification of representative data in g. Bound protein/input is shown. n = 3 (MTMR2) or 4 independent experiments. Data are mean ± SEM. See Source Data file for numerical source data and unprocessed blots.