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. 2015 Sep 24;16:17. doi: 10.1186/s12867-015-0045-5

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© Samra et al. 2015

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 1 — Purification of PMP1-assocaited proteins by RaPID. a Scheme of the tagged PMP1 mRNA and fusion MS2 binding protein. Lengths of the different mRNA regions are indicated. 423R indicates the position of the antisense oligo probe that was used in D (complementary to a region 380–423 nts downstream to the stop codon). The MS2 fusion protein includes an MS2 binding domain (MS2-CP, pink) fused in frame with GFP (green) and Streptavidin binding protein (SBP, yellow). b Key steps in RaPID. Protein lysate from cells expressing the tagged PMP1 and the MS2 fusion protein (input) is loaded on Streptavidin beads, subjected to extensive washes and bound material is eluted by addition of biotin. The eluted material is subjected to RNA analysis (panels d and e) or protein analysis (f, g). Ultimately, protein samples are subjected to mass spectroscopy to identify novel RNA binding proteins depicted as red circles. c PCR analysis for genomic DNA that was extracted either from cells with normal PMP1 (−MS2L) or cells that were subjected to MS2 loops insertion (+MS2L). PCR primers are from PMP1 promoter (forward) and 3′ UTR (reverse). DNA size markers are indicated to the left. d RNA was extracted from the indicated strains by the hot phenol method, and subjected to northern analysis with the indicated probes. RNA size markers are indicated to the left. e The indicated strains were subjected to RaPID, and RNA samples extracted from 5 % of the input lysate (input) or from half of the eluted material (elution) were subjected to northern analysis with the indicated probes. f Protein samples were collected along the RaPID protocol, and subjected to western analysis with the indicated antibodies. Samples from the following steps were analyzed: Input lysate (input), flow through (flow), last wash (wash) and eluted material (elution). Protein size markers are indicated to the left. Note that the Input lane seems split due to electrophoresis problem. g RaPID analysis was performed to cells either expressing untagged PMP1 (−MS2L) or tagged PMP1 (+MS2L). Protein samples from the Input or the Elution were subjected to western analysis with the indicated antibodies