Fig. 3.

Stathmin interacts with the NF-κB p65^RelA subunit in HTLV-1-transformed ATLL cells. (A) p65^RelA-Stathmin protein complexes were immunoprecipitated from precleared whole-cell lysates prepared from PMA-stimulated 293 HEK cells using Protein-G-agarose (20 ml of a 50% slurry) and monoclonal Anti-p65^RelA and rabbit polyclonal Anti-Stathmin/Op-18 antibodies. The relative input levels of Stathmin, p65^RelA, and Actin are shown in the upper panels. The precipitated bound p65^RelA and Stathmin proteins were resolved by SDS-PAGE and detected by immunoblotting (lower panels). UT, untreated cells. (B) The relative levels of the immunoprecipitated Stathmin (left) and p65^RelA (right) proteins in A were quantified by densitometry analysis of the immunoblot bands. (C) The formation of HTLV-1 Tax-induced p65^RelA-Stathmin immune-complexes is markedly diminished in cotransfected cells expressing increasing amounts (2.5 and 5.0 mg) of p30^II-GFP. The relative input levels of HTLV-1 Tax, p30^II-GFP, Stathmin, and Actin are shown (upper panels). The Stathmin/Op-18 protein, complexed with p65^RelA, was co-immunoprecipitated using Protein-G-agarose and an Anti-p65^RelA antibody and detected by immunoblotting (lower panel). (D) The relative levels of the immunoprecipitated Stathmin protein in C was quantified by densitometry. (E) p65^RelA-Stathmin protein complexes were immunoprecipitated from precleared extracts prepared from cultured/activated hu-PBMCs and the HTLV-1-transformed ATLL T-cell-lines, MJG11 and SLB1, using a monoclonal Anti-p65^RelA antibody. As a negative control, the Anti-p65^RelA antibody was heat-inactivated prior to its use in immunoprecipitation reactions. The relative input levels of Stathmin, p65^RelA, Tax, and Actin are shown in the upper panels. The precipitated Stathmin protein was resolved by SDS-PAGE and detected by immunoblotting using a rabbit polyclonal Anti-Stathmin/Op-18 antibody (lower panels). A non-specific band (ns) is also indicated. (F) The relative levels of immunoprecipitated Stathmin in E was quantified by densitometry analysis of the immunoblot bands. (G-J) siRNA-inhibition of Stathmin expression destabilizes the NF-κB p65^RelA subunit. HTLV-1-transformed SLB1 lymphoma T-cells were repeatedly transfected with 180 ng of a siRNA-stathmin oligonucleotide (#1 in G, or #2 in I) or non-specific RNA (nsRNA) as a negative control, and the Stathmin, p65^RelA, and HTLV-1 Tax proteins were detected by SDS-PAGE and immunoblotting. (H and J) The relative expression of Stathmin, p65^RelA and Tax in G (H) and I (J) was quantified by densitometry. All the data is representative of at least three independent experiments. The data in B, D, F, H and J represent the mean ± standard deviation (error bars).