Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-4

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>taining an inframe 3'-terminal myc-tag. Similar transfection efficiencies were controlled by measuring the luciferase activity in cell supernatants (not shown). Six hours following transfection cells were infected with the GFP expressing recombinant RSV at an MOI of 2 (+). As negative control, cells were also left uninfected (-). GFP expression analysis revealed similar infection efficiencies (data not shown). Equal amounts of protein were analysed in non-reducing Western blot analysis using a monoclonal antibody to the myc-tag 48 h after transfection

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-5

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>cate mutated consensus poly(A) signals. B) Western blot analysis of RSV-F protein levels after transfection of the indicated expression plasmids. An expression plasmid for EGFP was cotransfected. Undiluted lysates had equal protein content and a similar amount of fluorescent activity revealing constant transfection efficiency. Numbers above the lanes indicate the dilution at which the cell lysates were loaded on the gel

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-3

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>d in figure legend 3C. Plasmids pIFΔ2 and pIFΔ24 contain mutations in the second or the second and forth consensus poly(A) signal, respectively. B) Northern blot analysis of cytoplasmic RNA of 293T cells transfected with the indicated plasmids or HEp2 cells infected with RSV. Size separated RNA was stained with ethidiumbromide (EtBr) revealing non-degraded 18S and 28S ribosomal RNA bands (18S shown as representative). A DIG-labelled probe from the RSV-F ORF was used for hybridisation. C, D) Western blot analysis. 293T cells were cotransfected with an EGFP expression plasmid and the indicated RSV-F expression plasmids and analysed by Western blot using an anti-RSV-F monoclonal antibody. The lysate of pIFtransfected cells was diluted from 1:10 to 1:10, while the lysates from the other transfected cell were loaded at a 1:1 dilution. Similar transfection efficiencies were controlled for by measuring the fluorescence activity of cell lysates (data not shown)

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-0

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>ked by the human cytomegalovirus immediate early promoter/enhancer region (CMV) and the bovine growth hormone poly(A) signal (pA). Angled black arrows mark the transcriptional start point. The pIGvector contains intron A and flanking untranslated exonic regions E1 and E2 of the cytomegalovirus immediate early gene. In pIΔIGthe exon boundaries were precisely fused by deleting the intron. B) Northern blot analysis. Cells were cotransfected with the indicated VSV-G expression plasmids, a codon optimised HIV-1 expression plasmid (Hgp) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Poly(A) RNA was isolated from transfected cells and analysed by Northern blot with a probe spanning the transcribed region of the BGH poly(A) signal present on all VSV-G transcripts and the positive 5 kb HIV-1 transcript. C) Western blot analyses. Cells were cotransfected with the indicated VSV-G expression plasmids, an SIV expression plasmid (SgpΔ2) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Monoclonal antibodies to HIV-1 p24 capsid protein, which is cross reactive to SIV p27, or to VSV-G, respectively, were used for detection of the viral proteins in lysates of transfected cells

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-6

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p>ked by the human cytomegalovirus immediate early promoter/enhancer region (CMV) and the bovine growth hormone poly(A) signal (pA). Angled black arrows mark the transcriptional start point. The pIGvector contains intron A and flanking untranslated exonic regions E1 and E2 of the cytomegalovirus immediate early gene. In pIΔIGthe exon boundaries were precisely fused by deleting the intron. B) Northern blot analysis. Cells were cotransfected with the indicated VSV-G expression plasmids, a codon optimised HIV-1 expression plasmid (Hgp) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Poly(A) RNA was isolated from transfected cells and analysed by Northern blot with a probe spanning the transcribed region of the BGH poly(A) signal present on all VSV-G transcripts and the positive 5 kb HIV-1 transcript. C) Western blot analyses. Cells were cotransfected with the indicated VSV-G expression plasmids, an SIV expression plasmid (SgpΔ2) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Monoclonal antibodies to HIV-1 p24 capsid protein, which is cross reactive to SIV p27, or to VSV-G, respectively, were used for detection of the viral proteins in lysates of transfected cells

Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps-2

<p><b>Copyright information:</b></p><p>Taken from "Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps"</p><p>http://www.virologyj.com/content/4/1/51</p><p>Virology Journal 2007;4():51-51.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892776.</p><p></p> of primers used for the PCR analyses. The scale indicates the distance to the transcriptional start site. AATAAA: consensus signal for polyadenylation. B) Characterisation of splicing. 293T cells were transfected with pIF. Cytoplasmic RNA was isolated from transfected cells and reverse transcribed by oligo-dT priming (pIFcDNA). A PCR spanning the splice sites was performed with primers: 5'UTR-s and RSV-F-ia. The size of the PCR-products was compared to the size obtained in parallel PCR using pIΔIFand pIFplasmid-DNA (pDNA) as templates. HO: negative control. C) Characterisation of poly(A) signal usage. 293T cells were transfected with the indicated plasmids. Cytoplasmic RNA was isolated from transfected cells and reverse transcribed by priming with Oligo(dT)Add-a. As a control for DNA contamination, the reverse transcription reaction was also performed without the enzyme (-). The cDNA was amplified by PCR with primers 5' UTR-s and Oligo(dT)Add-a and the size of the PCR products was determined by agarose gel electrophoresis. D) PCR products from pIFtransfected cells were cloned and sequenced. The 3' end of the sequence obtained in 9 of 10 clones (RSV-F mRNA exp.) is shown aligned to the RSV-F sequence of the parental plasmid (RSV-F mRNA theor.)

The relative abundance of collagens and their subunits in supraspinatus tendon from control and pathologic female and male donors.

<p>Dot plots show biological replicates, means and standard error of the mean. (A) COL1A1, (B) COL1A2, (C) COL3A1, (D), COL5A1, (E) COL5A2, (F) COL6A1, (G) COL6A2, (H) COL6A3 and (I) COL14A1. (J) Floating bars showing minimum and maximum relative abundance of the subunits of collagen I with a line at the mean and (K) the relative abundance of the subunits of collagen VI. (L) The ratio of collagen III to I, average of ratios in individual runs within each group (duplicates per sample, n = 18 per group). *P<0.05, **P<0.01 significantly different from control.</p

Previously published studies confirming the disruption of COL1, COL6, FBN1, COMP and LTBP2 in torn and/or aged tendons.

<p>Previously published studies confirming the disruption of COL1, COL6, FBN1, COMP and LTBP2 in torn and/or aged tendons.</p

The interactions of all proteins which were significantly reduced in torn samples compared to control, created using the STRING database.

<p>The different line colours represent the different types of associations between the differentially modulated proteins, which include known interactions such as those identified in the curated database (light blue), and experimentally determined (purple). Other interactions identified were via text mining (green), co-expression (black) and protein homology (grey). Proteins are: COL1A1, Collagen 1(I); COL1A2, Collagen 2(I); COL6A1, Collagen 1(VI); COL6A2, Collagen 2(VI); FBLN1, Fibrillin-1; MFAP5, microfibrillar associated protein 5; COMP, Cartilage oligomeric protein; THSD4, Thrombospondin-4; CILP1, Cartilage intermediate layer protein 1; CILP2, Cartilage intermediate layer protein 2; TGFB1, Latent-transforming growth factor beta-binding protein 2; FBN1, Fibulin-1; TNXB, Tenascin-X; MYOC, Myocilin.</p

A quantitative label-free analysis of the extracellular proteome of human supraspinatus tendon reveals damage to the pericellular and elastic fibre niches in torn and aged tissue

<div><p>Tears of the human supraspinatus tendon are common and often cause painful and debilitating loss of function. Progressive failure of the tendon leading to structural abnormality and tearing is accompanied by numerous cellular and extra-cellular matrix (ECM) changes in the tendon tissue. This proteomics study aimed to compare torn and aged rotator cuff tissue to young and healthy tissue, and provide the first ECM inventory of human supraspinatus tendon generated using label-free quantitative LC-MS/MS. Employing two digestion protocols (trypsin and elastase), we analysed grain-sized tendon supraspinatus biopsies from older patients with torn tendons and from healthy, young controls. Our findings confirm measurable degradation of collagen fibrils and associated proteins in old and torn tendons, suggesting a significant loss of tissue organisation. A particularly marked reduction of cartilage oligomeric matrix protein (COMP) raises the possibility of using changes in levels of this glycoprotein as a marker of abnormal tissue, as previously suggested in horse models. Surprisingly, and despite using an elastase digestion for validation, elastin was not detected, suggesting that it is not highly abundant in human supraspinatus tendon as previously thought. Finally, we identified marked changes to the elastic fibre, fibrillin-rich niche and the pericellular matrix. Further investigation of these regions may yield other potential biomarkers and help to explain detrimental cellular processes associated with tendon ageing and tendinopathy.</p></div