Overexpression of nexilin suppresses IRS1/PI3K signaling in 3T3-L1 adipocytes.

<p><i>A</i>) 3T3-L1 adipocytes transduced with adenoviruses expressing FLAG-nexilin (ad-Nex) or GFP (Ad-GFP) were serum starved 72hrs post-infection and either left untreated or stimulated with increasing concentration of insulin. Cell lysates were subjected to Western blot analysis using the indicated antibodies. <i>B</i>) 3T3-L1 adipocytes transduced as in A) were starved or stimulated with 10 nM insulin and exposed to [³H] 2-deoxyglucose for 5 minutes. The reaction was ceased and the cells were harvested to assess the presence of [³H] within the cell through liquid scintillation counting. Data are means ± SE, n = 3 per group, one-way ANOVA (* <i>P</i><0.001).</p

Silencing of nexilin enhances IRS1/PI3K assembly.

<p>L6 myotubes were transfected with either scrambled (scr) or nexilin specific siRNA (si-nex) oligos. Serum depleted cells were stimulated with 100 nM insulin <i>A</i>) or 10 nM <i>B</i>) for the indicated times. IRS1 was immunoprecipitated from cell lysates and complexes probed with either 4G10, nexilin or p85α PI3K abs as indicated.</p

Silencing of nexilin enhances AKT activation and glucose uptake.

<p><i>A,B</i>) L6 myotubes transfected with either control or si-nex oligos and stimulated with increasing concentrations of insulin for 5 minutes. Whole cell lysates were resolved by SDS/PAGE and immunoblotted with the indicated antibodies. <i>C</i>) Glucose uptake in L6 myotubes. Serum-starved cells were incubated with 10 nM insulin for 20 minutes prior to exposure to [³H] 2-deoxyglucose for 5 minutes. Data are means ± SE, n = 3 per group, one-way ANOVA (* <i>P</i><0.05).</p

Nexilin is a novel binding partner of IRS1.

<p><i>A</i>) Nexilin selectively binds to IRS1 in L6 skeletal muscle cells. Serum starved L6 myotubes were left untreated or stimulated with 100 nM insulin for the indicated times. Cell lysates were immunoprecipitated (IP) with either IRS1 or IRS2 antibodies (abs) and subjected to western blot analysis with the indicated abs. WCL, whole cell lysates; <i>B</i>) Schematic representation of nexilin constructs. The isolated open reading frame of s-nexilin predicts a protein of 611 amino acids (aa) and consists of a central coiled-coil (CC) domain flanked by two F-actin binding domains (ABD). Nexilin-#2 is a truncated version containing the CC and second ABD domain (aa. 155–419) Nexilin-#3 consists of the second ABD domain (aa 240–410). Nexilin-#4 contains the N-terminal ABD and CC domains (aa 1–237). <i>C</i>) HEK293 cells were transfected with either pCMV5b vector (C), full length (FL) pCMV5b/Flag-nexilin construct or Flag-tagged nexilin-#2, #3 or #4 constructs. Cells were co-transfected with HA-IRS1. Left Panel, Lysates were immunoprecipitated with HA abs and blotted with either Flag or HA abs. Right Panel, Whole cell lysates (WCL) from transfected cells were immunoblotted with Flag abs showing expression of recombinant nexilin proteins.</p

Overexpression of Flag-nexilin inhibits localized PI3K activation in L6 Cells.

<p><i>A</i>) L6 myoblasts were transfected with Flag-nexilin or vector alone together with GRP1-PH-GFP cDNA. Following starvation, cells were stimulated with 100 nM insulin and then fixed, permeabilized and probed with anti-Flag antibodies followed by Cy3-conjugated donkey anti-mouse secondary abs (red). Cells were visualized for the presence of PIP3 accumulation in cell membranes using GRP1-PH-GFP. <i>B</i>) L6 cells were transfected with GRP1-PH-GFP and pretreated with Ly294002 (50 nM) prior to insulin stimulation and probed with anti-pAKT abs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055634#pone-0055634-g002" target="_blank">Figure 2</a>. <i>C</i>) L6 myoblasts transfected with Flag-nexilin or vector alone were treated with 100 nM insulin for the indicated times and then probed with anti-Flag abs and Cy5-conjugated secondary abs (green) and rhodamine-phalloidin (red).</p

Insulin-induced dissociation of IRS1/nexilin complex is dependent on F-actin remodeling.

<p>Left panel, IRS1 was immunoprecipitated from L6 myotubes that were either starved or insulin stimulated (100 nM) for the indicated times. Immune complexes were probed with anti-phosphotyrosine 4G10 or nexilin abs. WCL, whole cell lysates; Right Panel, Latrunculin B (20 µM, 20 min) or Jaspakinolide (2 µM, 30 min) treatment of L6 myotubes is without effect on the phosphorylation status of IRS1 but inhibits insulin-induced IRS1/nexilin disassembly.</p

Additional file 12: of Proteomic analysis of Medulloblastoma reveals functional biology with translational potential

Figure S8. Expression of HMAG1 isoforms in medulloblastoma tumors. a) Schematic representation of HMGA11 isoforms and Boxplots representing the mRNA expression levels for HMGA1 isoforms. b). Western blot of HMGA1 isoforms in the four medulloblastoma subgroups. Both HMAG1 isoforms are highly expressed in group 3 medulloblastoma. c) Kaplan–Meier survival curve shows that increased levels of HMGA1 are associated with poor survival in Group 3 Medulloblastoma. d) Expression level of HMGA1 is highly correlated with the expression of the oncogene MYC in Group 3 Medulloblastoma. (PDF 1.94 mb

Additional file 17: of Proteomic analysis of Medulloblastoma reveals functional biology with translational potential

Table S8. List of pathways representative for each medulloblastoma subgroup. (XLSX 72.6 kb

Additional file 11: of Proteomic analysis of Medulloblastoma reveals functional biology with translational potential

Figure S7. Expression of CALD1 isoforms in medulloblastoma tumors. The protein expression level of CALD1 isoforms in medulloblastoma subgroups is confirmed at the epigenetic (H3K27Ac Chip-seq) and mRNA level. a) Schematic representation of CALD1 isoforms. b) H3K27Ac Chip-seq genome tracks in medulloblastoma tumors. Active transcription region marks (H3K27Ac) are observed in the alternative transcription start site for the isoforms HeLa l-CaD I and II correlating with higher expression of these protein isoforms. c) Boxplots representing the mRNA expression levels for CALD1 isoforms. (PDF 2.2 mb

Additional file 7: of Proteomic analysis of Medulloblastoma reveals functional biology with translational potential

Figure S4. Copy number effect on mRNA and protein abundance on chromosome arm 17q. a) GRB2, LASP1, KPNB1 and ARHGDIA showed significant CNA-protein correlations (p < 0.05). The colors depict the range from low (white) to high (green) of copy-number, protein and mRNA abundance. Samples were ranked by copy number at each gene locus. b) ARHGDIA protein was found to be significantly overexpressed in group 4 tumors which frequently harbor 17q gains but not at the mRNA transcript level. Differences among the four subgroups were evaluated based on the Kruskal-Wallisrank-sum test. (PDF 916 kb