High-performance capillary electrophoresis for determining HIV-1 Tat protein in neurons

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Abstract

The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders. © 2011 Deshmane et al.

Figures

  • Figure 1. High performance capillary electrophoresis analysis of Tat protein. A. Electropherograms showing different concentrations of rTat in order to examine the sensitivity of the assay. B, C. Representative electropherograms comparing the amount of Tat in supernatant from infected cells or from lysates from uninfected neuronal SH-SY5Y cells to which the supernatant was added. doi:10.1371/journal.pone.0016148.g001
  • Figure 2. Measurement of Tat in primary human neurons (HN) and ability of rTat to activate HIV-1 gene expression. A. Representative electropherograms comparing the amount of Tat in supernatant from infected cells (red) or from extracts from uninfected (black) HN cells to which the supernatant was added. As a control, extracts were also prepared from rTat-treated cells (blue). B. Human microglia and SH-SY5Y cells were transfected with 0.5 mg of LTR-Luc alone and treated with different concentrations of rTat. C. SH-SY5Y cells expressing the LTR-luc-IRES-GFP were transfected with Tat expression plasmid, infected with Ad-null or Ad-Tat, or treated with rTat (1 pg/ml) or supernatant from infected cells. Cell extracts were prepared 24 h (microglia) or 48 h (SH-SY5Y) after transfection and luciferase and b-gal determined. All values were normalized against b-gal and represent mean of at least 3 experiments. doi:10.1371/journal.pone.0016148.g002
  • Figure 3. rTat cause dendritic retraction in SH-SY5Y. A. Phase contrast microscopy of SH-SY5Y cells either untreated or treated with rTat as indicated. The cells were stained with DAPI (blue) and anti-tubulin (red) to mark the nucleus and the processes, respectively. B. Tat-induced neurites retraction is time-dependent. Cell cultures were exposed to 1 pg/ml of rTat and assessed over 10 min. Values are expressed as percentage neurites retraction. P,0.01 vs control (untreated) (ANOVA, followed by Bonferroni test). Values represent mean (SD), (n = 15). C. Quantification of MAP-2 immunoreactivity revealed marked reduction (75%) in rTat-treated SH-SY5Y cells. D. Effect of rTat-treatment on SH-SY5Y neuronal death as assessed by trypan blue exclusion. doi:10.1371/journal.pone.0016148.g003

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APA

Deshmane, S. L., Mukerjee, R., Fan, S., & Sawaya, B. E. (2011). High-performance capillary electrophoresis for determining HIV-1 Tat protein in neurons. PLoS ONE, 6(1). https://doi.org/10.1371/journal.pone.0016148

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