23 research outputs found
Mass spectrometry analysis identifies PARP-1 as the CCR6DNP binding protein.
<p>Mass spectrometry analysis identifies PARP-1 as the CCR6DNP binding protein.</p
CCR6 expression in human cells treated with 3-aminobenzamide.
<p>(a) qPCR and (b) Western blot to show expression of CCR6 and PARP-1 in Jurkat T cells, HCT116 and Hela cells. Cells were treated with 0 mM (lane 1), 5 mM (lane 2) and 10 mM (lane 3) 3-AB for 72 hrs. For qPCR, data are shown as mean±s.d. (<i>n</i> = 3).</p
Building counter culture: the radical praxis of social movement milieux
This thesis falls into two parts. The first (chapters one to three) states the problematic of
the research, develops a critique of the dominant âsocial movementsâ literature as
unhelpful for understanding the counter culture and argues that the latter can more
effectively be theorised in terms of the implicit theory of social movement found within
agency-oriented Western Marxism and socialist feminism. This latter theory is developed as
an understanding of movement as direction, developing from the local rationalities of
everyday life through articulated but partial campaigns to a âmovement projectâ which
attempts to deploy such local rationalities to restructure the social whole. Within these
terms, it argues for an understanding of counter culture as a movement project from below
within disorganised capitalism. This mode of analysis is seen as that of a historical
sociology geared to the production of open concepts which can be used by participants to
theorise the context of their own choices.
The second part (chapters four to eight) theorises the issues involved in researching social
movements within this perspective, entailing the need to engage with tacit knowledge, to
thematise conflicts and collusion between researcher and participants. The findings
chapters use qualitative interviews from a Dublin movement milieu to develop an analysis,
grounded in participation, of the local rationalities of the counter culture. In this section
the key findings are a rationality of autonomy as self-development, which is shown to
underlie processes of distancing and problems of commitment, and a rationality of
radicalised reflexivity, which resolves the problem of institutionalisation through the
deployment of a wide range of âtechniques of the selfâ. The analysis attempts to locate this
reading within the life-histories of participants but also within the historical development
of the counter culture, examplifying the ability of the concepts developed in this thesis to
engage with the problems facing participants
The association of PARP-1 with CCR6DNP is sequence dependent and allele specific.
<p>(a) FREP+Western blot for PARP-1 with BamH I fractions. Lane 1: nuclear extract (NE) input, lane 2: NE with CCR6DNP/TG-beads, lane 3: NE with CCR6DNP/TG-beads with cold competitor (Free TG), and lane 4: NE with a non-specific DNA sequence (NS-beads). (b) To detect allele specificity, FREP was modified to cut directly with BamH I only, yielding a combined BamH I+EcoR I fraction, and SDS-PAGE was probed with anti-PARP-1. Anti-Ku86 antibody was used as the internal loading control to ensure comparable amounts of competitor DNA. CCR6DNP/TG-beads were incubated with NE and competed with equal amount of free competitors. Lane 1: no competitor; lane 2: free CCR6DNP/TG; lane 3: CCR6DNP/CG; lane 4: free CCR6DNP/CA; and lane 5: free non-specific DNA. Upper: Western blot; Lower: relative density from the Western blot. (c) ChIP assay with an anti-PARP-1 antibody on WT HCT116 cells and 5 mutants as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.g001" target="_blank">Fig 1</a> showing impaired binding of PARP-1 to the CCR6DNP. (d) ChIP assay with an anti-PARP-1 antibody on WT Jurkat T cells showing significant enrichment of the CCR6DNP fragments comparing to anti-IgG antibody. (e) Sequencing of the CCR6DNP fragments from the ChIP with WT HCT116 cells showing the significant enrichment of the T allele over the C allele by an anti-PARP-1 antibody. Results from (a), (b), (c), and (d) representative of 3 experiments. Data are shown as mean±s.d. (<i>n</i> = 3).</p
Characterization of mutated HCT116 clones generated by TALENs targeting the CCR6DNP.
<p>(a) Partial genomic arrangement of <i>CCR6</i> showing alternative transcripts CCR6-a and CCR6-b and the location of CCR6DNP. Only CCR-b could be detected in HCT116 and Jurkat T cells by real time PCR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.s003" target="_blank">S3 Fig</a>). (b) Sequence of the three alleles of CCR6DNP in 7 representative targeted HCT116 clones. The CCR6DNP is underlined. (c) and (d) Expression of CCR6 in the 7 targeted clones by qPCR and Western blot. For qPCR, data are shown as mean±s.d. (<i>n</i> = 3). Sequence, sequence trace and expression data for all 17 mutated HCT116 clones are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006292#pgen.1006292.s002" target="_blank">S2</a> Figs. Statistical significance in 1c reflects comparison to WT.</p
Flanking Restriction Enhanced Pulldown (FREP).
<p>(a) An 82 bp biotinylated DNA fragment is conjugated to streptavidin-coated magnetic Dynabeads (Invitrogen). This fragment is engineered to include a 31bp gene specific (âbaitâ) sequence (black box), flanked by restriction enzyme cleavage sites for BamH I proximally (blue box) and EcoR I distally (red box), as well as 20bp DNA fragments to allow PCR amplification of the whole unit. (b) DNA-beads are mixed with nuclear extract. A free 82 bp non-biotinylated DNA fragment can be included in the control reaction at this stage as a specific competitor. (c) Magnetic separation and wash to remove non-DNA binding proteins. (d) EcoR I digestion to release 3â DNA end-binding proteins, yielding the EcoR I fraction. (e) BamH I digestion to separate the sequence specific DNA binding proteins, BamH I fraction, from proteins that bind 5â DNA and Dynabeads. (f) Mass spectrometry (MS) to identify proteins remaining within the BamH I fraction.</p
FREP to identify the binding of PARP-1 with CCR6DNP.
<p>(a) Silver stain and (b) densitometry to show proteins pulled down from CCR6DNP/TG-beads mixed with nuclear extract from THP-1 cells. Lane 1, BamH I fraction; Lane 2, BamH I fraction where pulldown was performed in the presence of a 40x excess of competitor (free CCR6DNP/TG). Arrow indicates the specific band sent for mass spectrometry analysis.</p
CCR6 expression in PARP-1 knockdown human cells.
<p>HCT116 cells (a) and Jurkat T cells (b) by Western blot (left), and qPCR analysis on PARP-1 (middle) and on CCR6 (right). For Western blot, whole cell extract was isolated and separated on SDS-PAGE gel. Western blot was detected with mouse anti-human PARP-1, CCR6 and α-tubulin antibodies simultaneously. Lane 1: negative control for shRNA. Lane 2: shRNA treatment for HCT116 cells or siRNA treatment for Jurkat T cells. For qPCR, data are shown as mean±s.d. (<i>n</i> = 3).</p
P<i><sub>PCK1</sub></i>-driven OE of 11 <i>C. albicans</i> genes triggers pseudohyphal or hyphal growth.
<p><i>C. albicans</i> strains with integrated CIp10-P<i><sub>PCK1</sub></i>-GTW-TAPtag derivatives harbouring ORFs for the indicated genes were grown in SD (â) or YNB 2% casamino acids (+) for 6 h (Western Blot) or in YNB 2% casamino acids for 18 h (microscopy). Whole cell extracts of uninduced and induced cultures were separated by SDS-PAGE and probed with a peroxidase-coupled antibody allowing the detection of TAPtagged proteins in gluconeogenic conditions only. Proteins of interest are indicated by an arrow along with their deduced size. 18 h induced cultures were observed microscopically and revealed OE-associated pseudo-filamentation or filamentation. Note that the 5 ”m scale bar is different for photos of the two upper panels (GFP, <i>UME6</i>, <i>SUC1</i>, <i>SFL2</i>, and <i>GRF10</i>) and the rest of the figure.</p
P<i><sub>TET</sub></i>-driven OE screens confirm results obtained with the P<i><sub>PCK1</sub></i> promoter and reveal the role of other <i>C. albicans</i> genes in morphogenesis. A. P<i><sub>TET</sub></i>-driven OE of 21 genes promotes pseudo-filamentation or filamentation in liquid media.
<p><i>C. albicans</i> strains with integrated CIp10-P<i><sub>TET</sub></i>-GTW derivatives harbouring ORFs for the indicated genes were grown in YPD or YPD supplemented with 3 ”g.mL<b><sup>â</sup></b><sup>1</sup> ATc for 18 h. Both cultures were observed microscopically and revealed OE-associated pseudofilamentation or filamentation. Genes whose P<i><sub>PCK1</sub></i>-driven OE results in pseudo-hyphal or hyphal growth are shown on the left panel whereas others genes are placed on the right panel. OE of 15 genes showing the strongest phenotypes are represented, the other 6 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045912#pone.0045912.s002" target="_blank">Fig. S2</a>. Scale barâ=â5 ”m. <b>B. P</b><b><i><sub>TET</sub></i></b><b>-driven OE of 17 genes promotes filamentation on solid media.</b> Cultures of <i>C. albicans</i> wild-type strains with integrated CIp10-P<i><sub>TET</sub></i>-GTW derivatives harbouring ORFs for the indicated genes were spotted on YPD or YPD supplemented with 3 ”g.mL<b><sup>â</sup></b><sup>1</sup> ATc and were observed after 5 days of growth at 30°C. C. Overlap between the three morphogenesis screens. This venn diagram was obtained with the online software Gliffy (<a href="http://www.gliffy.com" target="_blank">http://www.gliffy.com</a>) and summarises results obtained in the screens performed with the P<i><sub>PCK1</sub></i> and P<i><sub>TET</sub></i> promoters. Circle size is proportional to the number of genes identified.</p