prl tk normalization plasmid  (Sangon Biotech)

Journal: Journal of Virology

Article Title: Human T-Cell Lymphotropic Virus Type 1 Transactivator Tax Exploits the XPB Subunit of TFIIH during Viral Transcription

doi: 10.1128/JVI.02171-19

Figure Lengend Snippet: XPB is involved in Tax-mediated LTR activation. (A) Effect of XPB overexpression on HTLV-1 LTR activation in Jurkat T cells. Cells were transfected with the LTR U3R-Luc and pRL-TK reporter plasmids along with the control (basal) or Tax (transactivation) plasmids and with the XPB-GFP or XPD-GFP construct and then treated with DMSO or with EPL or SP at 10 μM for 24 h. Upper panel: relative luciferase activity calculated by normalizing firefly/renilla ratios to that of DMSO-treated Tax-transfected cells (set to 1). Data are means ± SEM of three independent experiments performed in duplicate. Lower panel: protein expression levels detected by immunoblots. Level of each band was normalized to the level of DMSO-treated control cells (lane 1). Data are from one representative experiment out of three. (B) XPB-GFP interaction with Tax. Human 293T cells were transfected with a control plasmid or with the Tax plasmid together with a plasmid encoding the untagged or GFP-fused version of XPB. Total (lysates) or anti-Tax immunoprecipitated (IP) proteins were blotted with the anti-XPB or anti-Tax antibody. (C) Effect of untagged XPB overexpression on HTLV-1 LTR activation in Jurkat T cells. Cells were transfected with the LTR U3R-Luc and pRL-TK reporter plasmids along with the Tax and XPB constructs and then treated with DMSO or with EPL or SP at 10 μM for 24 h. Left panel: relative luciferase activity calculated by normalizing firefly/renilla ratios to that of DMSO-treated Tax-transfected cells (set to 1). Data are means ± SEM of three independent experiments performed in duplicate. Right panel: protein expression levels detected by immunoblots. Level of each band was normalized to the level of DMSO-treated Tax-transfected cells (lane 1). Data are from one representative experiment out of two. (D) Effect of XPB knockdown on LTR activation by Tax. RFP positive-sorted Jurkat T cells producing the control of XPB shRNA were induced (dox+) or not (dox-) and transfected with the U3R-Luc and pRL indicator plasmids with or without the Tax plasmid. Left panel: expression of Tax and XPB analyzed by immunoblots. Data correspond to one representative experiment out of three. Right panel: luciferase production. Data are means ± SEM of three independent experiments performed in duplicate. Statistical significance: *, P < 0.05; **, P < 0.01.

Article Snippet: The normalization plasmid pRL-TK (Renilla) was obtained from Promega.

Techniques: Activation Assay, Over Expression, Transfection, Construct, Luciferase, Activity Assay, Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, shRNA

Journal: Journal of Virology

Article Title: Human T-Cell Lymphotropic Virus Type 1 Transactivator Tax Exploits the XPB Subunit of TFIIH during Viral Transcription

doi: 10.1128/JVI.02171-19

Figure Lengend Snippet: The R-E-D domain of XPB is required for proviral LTR transactivation and endogenous Tax production. (A) Expression levels of LTR-regulating factors or TFIIH components in C8166 T cells treated with SP. (B) Capacity of XPB-GFP or XPD-GFP to rescue SP-mediated inhibition of LTR transactivation or endogenous Tax production. HTLV-1-transformed C8166 T cells were transfected with the LTR U3R-Luc and pRL-TK reporter plasmids and either the XPB-GFP or XPD-GFP construct and were then treated with DMSO or with EPL or SP at 10 μM for 48 h. Upper panel: relative luciferase activities calculated by normalizing firefly/renilla ratios to that of DMSO-treated control cells (bar 1). Data are means ± SEM of three independent experiments performed in duplicate. Lower panel: Tax, XPB-GFP, or XPD-GFP expression levels detected by immunoblotting. Data are from one representative experiment out of three. (C) Interaction of Tax with the XPB mutants. Upper panel: schematic representation of the XPB protein and position of the XPB mutations. Lower panel: interaction of Tax with wild-type XPB or XPB mutants. Human 293T cells were cotransfected with one of the XPB constructs along with a control or Tax expressor. Total proteins prepared 24 h posttransfection were analyzed before (lysates) or after the anti-Tax immunoprecipitation (IP Tax). Data correspond to one representative experiment out of two. (D) Capacity of wt or mutated XPB proteins to rescue SP-mediated inhibition of LTR transactivation or endogenous Tax production. HTLV-1-transformed C8166 T cells were transfected with the LTR U3R-Luc and pRL-TK reporter plasmids and one of the XPB constructs and were then treated with DMSO or with EPL or SP at 10 μM for 48 h. Upper panel: relative luciferase activity calculated by normalizing firefly/renilla ratios to that of DMSO-treated control cells (bar 1). Data are means ± SEM of five independent experiments performed in duplicate. Lower panel: Tax and XPB expression levels detected by immunoblotting. Data are from one representative experiment out of three. Statistical significance: ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: The normalization plasmid pRL-TK (Renilla) was obtained from Promega.

Techniques: Expressing, Inhibition, Transformation Assay, Transfection, Construct, Luciferase, Western Blot, Immunoprecipitation, Activity Assay