Recombinant hiv 1 tat clade b protein

Puromycin was used for selection of the cells transduced with the Cav-1 shRNA lentiviral vectors because of the lentiviral vector pHBLV-U6-Scramble-ZsGreen-Puro having Pac gene. Recombinant HIV-1 Tat clade-B protein (amino acids 1 to 86) was purchased from ProSpec (Rehovot, Israel). The effects of puromycin and HIV-1 Tat on the viability of HBEC-5i cells were assessed by a Cell Counting Kit-8 assay (Biotech, Beijing, China). Briefly, a density of 1 × 10⁴ cells/well HBEC-5i cells was seeded onto 96-well plates. After 12 h, HBEC-5i cells were treated with puromycin at 0, 0.5, 1, 1.5, 2, 2.5, or 3 μg/mL or Tat at 0, 0.5, 1.0, 1.5, 2, 2.5, or 3 μg/mL for 24 h. Ten microliters of the CCK8 solution was added to each well of the plate and incubated at 37°C for 1 h. The absorbance at 450 nm was recorded using an iMark microplate reader (Bio-Rad, Hercules, CA) at 24 h.

Recombinant HIV-1 Tat clade B protein (Prospec, Rehovot, Israel), produced in Escherichia coli, is formed of a single, nonglycosylated, polypeptide chain containing 86 amino acids encoded by exons, with a molecular mass of 14 kDa. The amino acid sequence is as follows: MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFIT KALGISYGRK KRRQRRRPPQ GSQTHQVSLS KQPTSQSRGD PTGPKE. It is recommended that lyophilized HIV-1 Tat is stored desiccated below −18°C and reconstituted in sterile 18 MΩ-cm H₂O.

Human cerebral microvascular endothelial cells (HBEC-5i) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured on 0.1% gelatin solution (ATCC)-coated flasks in DMEM:F12 medium (ATCC) supplemented with 10% foetal bovine serum (Gibco/Thermo Fisher, Waltham, MA, USA), 40 μg/mL endothelial growth supplement (ECGS, ATCC), and 1% penicillin-streptomycin (Beyotime, Shanghai) according to the manufacturer's instructions. HBEC-5i cells were incubated at 37°C in a humidified atmosphere of 5% CO₂.
Recombinant HIV-1 Tat clade-B protein (amino acids 1 to 86) was purchased from Prospec (Rehovot, Israel). The previous literature indicated that concentrations of Tat in HIV-infected patients could reach the range of 0.5 μg/mL of serum [24 (link)], so this concentration was used in subsequent experiments. Controls consisted of cells treated with 0.02% DMSO or heat-inactivated Tat (1 μg/mL). Before exposure to 1 μg/mL HIV-1 Tat for 12 or 24 h, confluent HBEC-5i cells were pretreated with 5 μmol/L FTS for 3 h and FTS was retained in the serum-free cell culture medium during Tat treatment as previously described [25 (link)].