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Blood, Vol. 114, Issue 1, 85-94, July 2, 2009
HIV-1–infected dendritic cells show 2 phases of gene expression changes, with lysosomal enzyme activity decreased during the second phase
Blood Harman et al.
114: 85
Supplemental materials for: Harman et al
Western blot analysis of viable and AT-2 inactivated HIV-1 viral lysates
Virions from viable and AT-2 treated HIV-1BaL stocks were lysed with 10 µl of Bermann lysis buffer (1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, complete mini EDTA-free) (Roche) and loaded onto a 12% SDS-PAGE gel. Proteins were blotted onto PVDF membrane (Bio-rad, Hercules, CA) and probed with pooled human HIV-1 positive serum.
Files in this Data Supplement:
- Table S1. Primer sequences for QPCR (PDF, 48.9 KB)
- Table S2. Comparison of B value and absolute fold change as a determinate for reliability of microarray data (PDF, 22.9 KB) -
The table shows the sensitivity and specificity associated with values illustrated in the ROC curves (Fig. 2) comparing gene expression data generated by cDNA microarrays with qPCR. Bold type indicates B value cut points that were chosen by which to filter the microarray derived differential expression data. Sen = sensitivity, Spec = specificity.
- Table S3. Up regulated genes encoding proteins associated with the endosome and GTPase activity in response to viable and AT2 inactivated HIV (PDF, 64.8 KB) -
The table shows the gene bank accession number symbol and name, the fold change increase in its expression (FC) and B Value (B), for all endosomal and GTPase associated genes up regulated in MDDCs response to treatment with viable or AT-2 inactivated HIV.
- Figure S1. Quantification of high titre purified HIV-1 stocks (JPG, 247 KB)
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HIV-1 was concentrated from supernatants derived from infected PM1 cells using tangential filter concentration. Half the virus stock was treated with aldrithriol-2 (AT-2) and both stocks then purified using Optiprep gradient centrifugation. (A) Determination of relative virion concentrations in AT-2 treated and viable HIV-1 stocks. The relative quantities of HIV virions in each virus stock was by determined by comparing the relative amount of p24 by western blot/densitometry and ELISA. (B) Diagrammatic representation of the combinations of fluorescently labeled cDNAs that were hybridised to the 8K microarrays. (C) QPCR based HIV infectivity assay. 1 × 106 day 6 MDDCs were mock treated or treated with either the viable HIV or AT-2 inactivated HIV stock at MOI 10 (or equivalent AT-2 HIV dose) or with monomeric gp120 at 50ng/ml. The percentage of infected cells was measured at 6, 24 and 48 hours by QPCR using primers directed towards the HIV-1 LTR-gag region, assuming that cells are not infected by greater than 1 copy of DNA. The data from four experiments are shown with standard error bars.
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