Recombinant Tax1 and Tax2A (subtype A) proteins were purified as described recently in detail [24, 25]. Truncated Tax2A/NH2term-His tagged sequence aa 1–198 (Tax2A/1–198) (MRGSHHHHHHGS AHFPGFGQSL LYGYPVYVFG DCVQADWCPV SGGLCSTRLH RHALLATCPE HQL TWDPIDG RVVSSPLQYL IPRLPSFPTQ RTSRTLKVLT PPTTPVSPKV PPAFFQSMRK HTPYRNGCLE

PTLGDQLPSL AFPEPGLRPQ NIYTTWGKTV VCLYLYQLSP PMTWPLIPHV IFCHPRQLGA FLTKVPLKRL EELLYKM LDLQPSLIS) and truncated Tax2A/COOHterm-His tagged sequence aa 135–331 (Tax2A/135–331) (MRGSHHHHHHGS EPGLRPQNIY TTWGKTVVCL YLYQLSPPMT WPLIPHVIFC HPRQLGAFLT KVPLKRLEEL LYKMFLHTGT VIVLPEDDLP TTMFQPVRAP CIQTAWCTGL LPYHSILTTP https://www.selleckchem.com/products/LBH-589.html GLIWTFNDGS PMISGPCPKA GQPSLVVQSS LLIFEKFQTK AFHPSYLLSH QLIQYSSFHN LHLLFDEYTN IPVSILFNKE EADDNGD LDLQPSLIS) fragments of Tax2A protein containing NF-κB domains [28, 29] were subcloned from PET29a-Tax2-H6 [30] to pQE-30 (Amp-resistant) vector and transformed into the Esherichia coli BL21(DE3) strain (subcloning generation and protein production serviced by Promab Biotechnologies, Inc., Richmond, RXDX-106 ic50 CA, USA). An extract was prepared in an identical manner from E. coli

cells containing the empty vector for use as a mock control. Determination of protein concentrations was performed using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Endotoxin concentration for all protein recombinants at the concentration (100 pM) used in the in-vitro experiments were found to be endotoxin-free, as determined Dichloromethane dehalogenase by the limulus amoebocyte lysate test (E-TOXATE; Sigma) [24]. Recombinant replication-deficient adenoviruses expressing Tax2B (subtype B) or green fluorescent protein (GFP), used to control the efficiency of transduction (Ad-Tax2B or Ad-GFP, respectively) [31], were propagated and titrated as described recently [25]. The recombinant adenovirus containing the dominant negative mutant of IκBα with serine to alanine substitutions at amino acids 32 and 36, and therefore resistant to phosphorylation-induced degradation (a NF-κB super-repressor

designated NF-κB/SR), was obtained commercially (Vector Biolabs, Philadelphia, PA, USA). In this study the two major subtypes of HTLV-2 Tax, Tax2A (expressed as recombinant protein) and Tax2B (recombinant adenovirus) were assessed to determine whether both Tax2 subtypes were able to induce the production of CC-chemokines in peripheral blood mononuclear cells. PBMCs (1 × 106/ml) in complete RPMI medium were treated with extracellular Tax (recombinant Tax1 and Tax2A) proteins at 100 pM for 1, 2, 3, 6, 12 and 24 h to determine CC-chemokine production, and for 1 and 2 h for the determination of canonical NF-κB pathway activation. Mock-treated and untreated controls were used in all experiments.