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Results: 5

1.
Figure 5

Figure 5. Subcellular localization of the identified hits within a model cell.. From: Brain Transcriptome-Wide Screen for HIV-1 Nef Protein Interaction Partners Reveals Various Membrane-Associated Proteins.

Overview of the subcellular localization of the interaction partners of Nef identified via the split-ubiquitin based Y2H system based on the data given in Table S1. Marked in magenta are the proteins studied in more detail.

Ellen C. Kammula, et al. PLoS One. 2012;7(12):e51578.
2.
Figure 3

Figure 3. Analysis of GPM6B-induced outgrowth in Cos-7 cells.. From: Brain Transcriptome-Wide Screen for HIV-1 Nef Protein Interaction Partners Reveals Various Membrane-Associated Proteins.

Cells were treated as described in Figure 2. For enhanced visualization of the membrane extensions, magnifications of the boxed areas are depicted to the right of each picture. Representative images of cells transfected with pGFP-GPM6B without (A) or with pNef-DsRed (B) or pDsRed (C) cotransfection are shown. Scale bar: 10 µm.

Ellen C. Kammula, et al. PLoS One. 2012;7(12):e51578.
3.
Figure 2

Figure 2. Analysis of subcellular localization of BAP31, CD320/TCblR, CLDN10 and GPM6B with and without Nef.. From: Brain Transcriptome-Wide Screen for HIV-1 Nef Protein Interaction Partners Reveals Various Membrane-Associated Proteins.

A. Confocal microscopy analysis of Cos-7 cells coexpressing GFP fusions of BAP31, CD320/TCblR, CLDN10 or GPM6B with Nef-DsRed. Cos-7 cells were transiently cotransfected with pNef-DsRed and pGFP-BAP31, pGFP-CD320/TCblR, pGFP-CLDN10 or pGFP-GPM6B and fixed 24 h posttransfection. Single images from the red (Nef-DsRed) and green (GFP-prey) channels were overlaid in the merged image. Yellow regions represent colocalization, details are given in the text. Arrows point out BAP31 accumulations and distinct areas of CLDN10 and Nef overlay. Scale bar: 10 µm. B. Negative controls for the colocalization images in Figure A. Confocal microscopy analysis of Cos-7 cells coexpressing GFP fusions of BAP31, CD320/TCblR, CLDN10 or GPM6B with DsRed. Cos-7 cells were transiently cotransfected with pDsRed and pGFP-BAP31, pGFP-CD320/TCblR, pGFP-CLDN10 or pGFP-GPM6B and fixed 24 h posttransfection. Single images from the red (DsRed) and green (GFP-prey) channels were overlaid in the merged image. The respective scatter grams, as well as the images and scatter grams from cotransfections with the pDsRed control vector are given.

Ellen C. Kammula, et al. PLoS One. 2012;7(12):e51578.
4.
Figure 1

Figure 1. Schematic of the Y2H screen and coimmunoprecipitation of selected preys show interaction with Nef.. From: Brain Transcriptome-Wide Screen for HIV-1 Nef Protein Interaction Partners Reveals Various Membrane-Associated Proteins.

A. Schematic presentation of the split-ubiquitin Y2H screen with membrane-anchored Nef as bait. Wild type Nef myristoylation is replaced by the Ost4p transmembrane anchor and amino acids 39–76 of yeast ubiquitin (Cub) linked to the LexA-VP16 transcription factor carboxy-terminally to Nef. A human adult brain cDNA library (Dualsystems Biotech AG) cloned in pPR3-N expressing the cDNAs as fusions carboxy-terminally of amino acids 1–38 of yeast ubiquitin (Nub) and an HA-tag. Upon binding of the bait and prey both parts of the ubiquitin come together and are cleaved by the protease to activate reporter genes. If no interaction with the Nef bait protein is possible, the ubiquitin subunits stay apart and no reporter genes in the nucleus are turned on. B. Coimmunoprecipitation (CoIP) of Nef and preys. Yeast cell lysate proteins from different transfected cultures as well as a non-transfected control (NMY51) as indicated at the top were immunoprecipitated (IP) either with anti-HA or anti-LexA antibodies. For negative controls, cells were alternatively transfected with a bait expression vector coding for the SV40 large T antigen (largeT) instead of Nef. The resulting immunoprecipitates were electrophoretically separated, blotted (WB) on a PVDF-membrane as indicated at the right and detected with anti-Myc or anti-HA antibodies. The approximate molecular weights of the proteins are shown. Two additional experiments gave similar results.

Ellen C. Kammula, et al. PLoS One. 2012;7(12):e51578.
5.
Figure 4

Figure 4. Sequence analysis of Nef binding site and in vitro binding studies of GPM6B and Nef.. From: Brain Transcriptome-Wide Screen for HIV-1 Nef Protein Interaction Partners Reveals Various Membrane-Associated Proteins.

A. Amino acid sequence of the Nef binding motif of CD4. B. Clustal W [70] based alignment of the cytoplasmic loops of GPM6B, GPM6A and PLP1. Note, for GPM6B, GPM6A and PLP-DM20/PLP1 isoform 2, the sequences of the complete cytoplasmic loop regions are shown. Additionally, positions of the indicated proteolipid proteins with homologies to a sequence region in the cytoplasmic tail of T-cell surface glycoprotein CD4 (shown at the top) that includes the core Nef binding motif of CD4 [20], [21] are highlighted. Positions identical to the respective CD4 residue are shaded black, those with high similarity are shaded grey and those with lower similarity are boxed. The indicated residue numbers show the beginning and the end of the amino-terminally fluoresceinylated GPM6B and PLP1 peptides used for the Nef binding studies shown in D. Numbering of GPM6B is based on UniProtKB entry Q13491-1 throughout this figure. C. Sequence of the cytoplasmatic portion of CD320/TCblR, homologies to CD4 indicated as described above. D. Fluorescence titration of 0.5 µM of fluoresceinyl-labeled peptides, GPM6B112–127, GPM6BAA, or PLP193–108 with recombinant HIV-1SF2 Nef2–210 protein (prepared as described in [68]). The fluorescence signals are shown as a function of the Nef2–210 protein concentration. Values result from the fluorescence of the peptides in the presence of the indicated concentration of Nef2–210 in comparison with a buffer control titration. Assuming a simple bimolar interaction between the peptide and Nef2–210, the data were described by a model based solely on the law of mass action which accounts for ligand depletion [71]. Nonlinear curve fitting of the model to the fluorescence data (lines) yielded dissociation constants of 0.64±0.06 µM for GPM6B112–127 and of 0.71±0.03 µM for PLP193–108.

Ellen C. Kammula, et al. PLoS One. 2012;7(12):e51578.

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