Figure 2
Figure 2. Repression of wt P2 promoter activity by Tax. (A) Fresh CD4+ cells were transfected with HTLV-1 provirus pACH-wtTax DNA using Amaxa Kit. Cells were then cultured in AIM-V media with 10% fetal bovine serum, IL-2 (100 U/mL), and PHA (1 μg/mL). Cell were collected on day 0 (d0), day 3 (d3), and day 7 (d7) after transfection, and cell lysates were subjected to Western blot analysis. SHP-1 (sc-287) and β-tubulin (sc-9104) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Tax (Tab172) was from NIH-ARRRP. Jurkat E6-1 and MT2 cell line were used as the positive and negative of SHP-1 expression. (B) Jurkat-LT cells were transfected with 500 ng Wt-P2-Luc plasmid and different amounts of Tax plasmid (0, 10, 50, 200, 1000 ng). The effect of Tax on SHP-1 P2 promoter activity was measured by luciferase assay as described in “Transient transfection and luciferase assay.” Effect of E-box and NF-κB mutations on P2 promoter. Two E-box motifs and 2 NF-κB motifs were predicted in the SHP-1 LC-P2 promoter (Figure 1C). Site-specific mutants were generated to analyze the effects of these sites on the promoter activity. The sequences are shown below: NF-κB1, 5′-CAAGTGA/TGTTCCCCCAAGGG-3′; NF-κB2, 5′-CCTCTCCGGAAGCCCC/TCAGG-3′; Ebox1, AGAAGTAC/TAAGTGAGTTCCC; Ebox2, GGAGCTGCATCT/AGAGGCTTA. The italic sequences are the predicted wild-type motifs, and the bold letters represent the mutated nucleotides. Mu-Ebox1 + 2 and mu-NF-κB1 + 2 represent double mutations in E-boxes or NF-κB motifs, respectively. Asterisks indicate that the differences in values are statistically significant when compared with the values of the corresponding samples without Tax transfection. (C) Luciferase assay was performed to analyze the effect of promoter mutations described in panel B. Wild-type, large or small core SHP-1 P2 promoter plasmids, or the SHP-1 LC-P2 promoter mutant plasmids (0.5 μg) were transfected into Jurkat-LT cells in the presence or absence of 0.2 μg Tax plasmid. (D) Involvement of CREB and RelA in SHP-1 P2 basal promoter regulation was examined by luciferase assay of transiently cotransfected Jurkat-LT cell lysates. CREB-dependent HTLV-1-luc reporter and NF-κB–dependent 3xκb-luc reporter were similarly cotransfected with CREB or RelA encoding plasmids and the cell lysates were used as controls in the luciferase assay. Error bars represent means plus or minus SD.

Repression of wt P2 promoter activity by Tax. (A) Fresh CD4+ cells were transfected with HTLV-1 provirus pACH-wtTax DNA using Amaxa Kit. Cells were then cultured in AIM-V media with 10% fetal bovine serum, IL-2 (100 U/mL), and PHA (1 μg/mL). Cell were collected on day 0 (d0), day 3 (d3), and day 7 (d7) after transfection, and cell lysates were subjected to Western blot analysis. SHP-1 (sc-287) and β-tubulin (sc-9104) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Tax (Tab172) was from NIH-ARRRP. Jurkat E6-1 and MT2 cell line were used as the positive and negative of SHP-1 expression. (B) Jurkat-LT cells were transfected with 500 ng Wt-P2-Luc plasmid and different amounts of Tax plasmid (0, 10, 50, 200, 1000 ng). The effect of Tax on SHP-1 P2 promoter activity was measured by luciferase assay as described in “Transient transfection and luciferase assay.” Effect of E-box and NF-κB mutations on P2 promoter. Two E-box motifs and 2 NF-κB motifs were predicted in the SHP-1 LC-P2 promoter (Figure 1C). Site-specific mutants were generated to analyze the effects of these sites on the promoter activity. The sequences are shown below: NF-κB1, 5′-CAAGTGA/TGTTCCCCCAAGGG-3′; NF-κB2, 5′-CCTCTCCGGAAGCCCC/TCAGG-3′; Ebox1, AGAAGTAC/TAAGTGAGTTCCC; Ebox2, GGAGCTGCATCT/AGAGGCTTA. The italic sequences are the predicted wild-type motifs, and the bold letters represent the mutated nucleotides. Mu-Ebox1 + 2 and mu-NF-κB1 + 2 represent double mutations in E-boxes or NF-κB motifs, respectively. Asterisks indicate that the differences in values are statistically significant when compared with the values of the corresponding samples without Tax transfection. (C) Luciferase assay was performed to analyze the effect of promoter mutations described in panel B. Wild-type, large or small core SHP-1 P2 promoter plasmids, or the SHP-1 LC-P2 promoter mutant plasmids (0.5 μg) were transfected into Jurkat-LT cells in the presence or absence of 0.2 μg Tax plasmid. (D) Involvement of CREB and RelA in SHP-1 P2 basal promoter regulation was examined by luciferase assay of transiently cotransfected Jurkat-LT cell lysates. CREB-dependent HTLV-1-luc reporter and NF-κB–dependent 3xκb-luc reporter were similarly cotransfected with CREB or RelA encoding plasmids and the cell lysates were used as controls in the luciferase assay. Error bars represent means plus or minus SD.

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