Figure 2
Figure 2. Tax and p30 increase β-catenin transcriptional activity through AKT and/or p38 pathways. (A) Western blot analysis of phosphorylated GSK3-β (Ser9) expression from total cellular extracts of R.PBMCs, Jurkat, and HTLV-I cell lines. Extracts were normalized to actin expression. (B) Western blot analysis of total cellular extract for phosphorylated GSK3-β (Ser9) from 293T cells transfected with pc-Tax (0.5 µg) or PMH-p30 (2.0 µg). Extracts were analyzed 48 hours after transfection and normalized to actin expression. Western blots were stripped and reprobed for GSK3-β expression. Tax and HA-p30 expression were demonstrated in the transfected lysates. (C) PCR analysis of Tax and p30 expression in HTLV-I cell lines. Jurkat cDNA was used as a negative control. (D) pc-Tax (0.1, 0.2, and 0.5 µg) and PMH-p30 (0.5, 1.0, and 2.0 µg) were transfected into 293T cells along with the TOPflash luciferase reporter plasmid. Cellular extracts were used in luciferase assays to measure the level of β-catenin activity. All experiments were performed in duplicate, and values represent the average reading normalized to Renilla luciferase or protein concentration. Error bars represent the population standard deviation for each sample. (E) 293T cells were transfected with or without 1.0 µg PMH-p30, along with 0.1 µg of wild-type or mutant Tax and the TOPflash reporter plasmid, and analyzed as in (D). (F) 293T cells were transfected as in (B) and analyzed for β-catenin expression by real-time PCR. Real-time PCR was performed in duplicate, and samples were normalized to GAPDH expression. Fold change was calculated by comparing values with PMH-transfected cell–normalized gene expression. (G) 293T cells were transfected as in (D). Total extracts were analyzed 48 hours after transfection and normalized to actin expression. Tax and HA-p30 expression were demonstrated in the transfected lysates. (H-I) 293T cells were transfected in duplicate with 0.1 µg pc-Tax (H) or 1.0 µg PMH-p30 (I), along with 1.0 µg of dominant-negative AKT (DN AKT) or dominant-negative p38 (DN p38), and the TOPflash reporter plasmid. Values represent the average reading normalized to protein concentration. Error bars represent the population standard deviation for each sample.

Tax and p30 increase β-catenin transcriptional activity through AKT and/or p38 pathways. (A) Western blot analysis of phosphorylated GSK3-β (Ser9) expression from total cellular extracts of R.PBMCs, Jurkat, and HTLV-I cell lines. Extracts were normalized to actin expression. (B) Western blot analysis of total cellular extract for phosphorylated GSK3-β (Ser9) from 293T cells transfected with pc-Tax (0.5 µg) or PMH-p30 (2.0 µg). Extracts were analyzed 48 hours after transfection and normalized to actin expression. Western blots were stripped and reprobed for GSK3-β expression. Tax and HA-p30 expression were demonstrated in the transfected lysates. (C) PCR analysis of Tax and p30 expression in HTLV-I cell lines. Jurkat cDNA was used as a negative control. (D) pc-Tax (0.1, 0.2, and 0.5 µg) and PMH-p30 (0.5, 1.0, and 2.0 µg) were transfected into 293T cells along with the TOPflash luciferase reporter plasmid. Cellular extracts were used in luciferase assays to measure the level of β-catenin activity. All experiments were performed in duplicate, and values represent the average reading normalized to Renilla luciferase or protein concentration. Error bars represent the population standard deviation for each sample. (E) 293T cells were transfected with or without 1.0 µg PMH-p30, along with 0.1 µg of wild-type or mutant Tax and the TOPflash reporter plasmid, and analyzed as in (D). (F) 293T cells were transfected as in (B) and analyzed for β-catenin expression by real-time PCR. Real-time PCR was performed in duplicate, and samples were normalized to GAPDH expression. Fold change was calculated by comparing values with PMH-transfected cell–normalized gene expression. (G) 293T cells were transfected as in (D). Total extracts were analyzed 48 hours after transfection and normalized to actin expression. Tax and HA-p30 expression were demonstrated in the transfected lysates. (H-I) 293T cells were transfected in duplicate with 0.1 µg pc-Tax (H) or 1.0 µg PMH-p30 (I), along with 1.0 µg of dominant-negative AKT (DN AKT) or dominant-negative p38 (DN p38), and the TOPflash reporter plasmid. Values represent the average reading normalized to protein concentration. Error bars represent the population standard deviation for each sample.

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