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Figure 6 | Retrovirology

Figure 6

From: Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat protein

Figure 6

Triptolide reduces Tat protein stability by enhancing proteasomal degradation of Tat. (A) The effect of triptolide on Tat mRNA levels. HeLa cells were transfected with pEF1-Tat in the presence of increasing concentrations of triptolide. Cells were harvested 48 h post-transfection. The steady-state mRNA levels of Tat and GAPDH were examined using semi-quantitative reverse transcription PCR analysis. RT, reverse transcriptase. (B) The effect of triptolide on Tat protein degradation. HeLa cells were transfected with pEF1-FLAG-Tat in the presence of 5 nM triptolide or vehicle (DMSO). At 28 h post-transfection, cells were treated with 20 μg/mL of CHX to arrest protein synthesis. At 0, 1, 2, 3 h post-treatment, cells were harvested to determine protein expression levels using Western blot analysis. (C) The band intensities of FLAG-Tat in panel B were measured, and their natural log values were plotted as a function of time. Results were presented as the mean plus the standard deviations (n = 3). (D) Proteasome inhibition restores Tat protein expression. HeLa cells were transfected with pEF1-FLAG-Tat in the presence or absence of 5 nM triptolide. At 48 h post-transfection, cells were treated with 20 μM of MG132, and cultured for an additional 3 h before harvest. Protein expression levels were determined by Western blot analysis.

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