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Blood, Vol. 109, Issue 3, 1051-1060, February 1, 2007
Human T-cell leukemia virus type-1 Tax oncoprotein regulates G-protein signaling
Blood Twizere et al.
109: 1051
Supplemental materials for: Twizere et al, Blood, Vol 109, Issue 3, 1051-1060
Files in this Data Supplement:
- Figure S1. HTLV-1 Tax interacts with Gβγ complex but not with Gα subunit of heterotrimeric G proteins (PDF, 413 KB) -
The G 2 4 complex and G 15 protein were synthesized in vitro using rabbit reticulocyte lysates in the presence of 35S-labeled methionine and cysteine (A, top). Ten microliters lysates were then incubated for 3 hours with GST–Tax-1 produced in bacteria and bound to glutathione-Sepharose beads. After incubation, the protein complexes were separated by SDS-PAGE, stained with Coomassie blue (A, bottom), and visualized by autoradiography (A, middle). (B) Five microliters G24 complex and increasing amounts of G15 were mixed, incubated for 4 hours at 4°C, and pulled down using GST–Tax-1 protein, as described.
- Figure S2. Colocalization between HTLV-1 Tax and Gβ5 (PDF, 2.61 MB) -
HeLa cells were transfected with Flag-G5 (A) or Flag-G5 and HTLV-1 Tax expressing constructs (B). Twenty-four hours after transfection, cells were fixed, permeabilized, and stained with anti-Flag or anti–Tax-1 antibodies and Alexa 546 or fluorescein-conjugated secondary antibodies. Cells were labeled with TOPRO-3 and analyzed under a confocal microscope.
- Figure S3. Colocalization between BLV Tax and Gβ2 (PDF, 3.09 MB) -
HeLa cells were cotransfected with TaxBLV and Flag-G2 (A) or TaxHTLV-2 and Flag-G2 (B) expressing constructs. Twenty-four hours after transfection, cells were fixed, permeabilized, and stained with anti-Flag and specific anti-Tax antibodies, followed by Alexa 546 and fluorescein-conjugated secondary antibodies. Cells were labeled with TOPRO-3 and analyzed under a confocal microscope.
- Figure S4. Intracellular cascade activation and migration towards SDF-1 of T-cells over-expressing Gβ2 or HTLV-1 Tax proteins (PDF, 789 KB) -
(A) Tesi cells were transduced by a lentiviral system to express G-protein 2. Forty-eight hours after infection, a fraction of transduced cells was lysed and analyzed by Western blotting using an anti-G2 antibody or antiactin antibodies. (B) Tesi cells overexpressing G2 (Tesi-Lenti-G2) and control Tesi cells were stimulated for 5 minutes by 30 nM SDF-1. Cells were then lysed, and ERK1/2 activation was measured by Western blotting using an anti–phospho-p42/44 monoclonal antibody (top). (C) Tesi-Lenti-G2 and control Tesi cell migration assays were performed using Transwell culture chambers (5-µm pore size). Lower wells were filled with 500 µL medium (RPMI 1640, 1% FCS) containing 30 nM SDF-1. Cells suspended in 100 µL medium were loaded into the upper wells. After incubation for 2 hours at 37°C, the cells that had migrated to the lower wells were counted and are shown as percentages of the input cells. (D) MT4 cells were stimulated for 5 minutes by 30 nM SDF-1, 30 nM MCP-1, or 50 nM RANTES, and ERK1/2 activation was measured as described with an anti–phospho-p42/44 monoclonal antibody (top). The same blot was stripped, and immunoblotting performed using anti–Tax-1 antibody (bottom). (E) MT4 cell migration assays were performed using Transwell culture chambers (5-µm pore size) as described.
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