Fig. 2. hCAF1 and hPOP2 act as bridges between hCCR4 and BTG2. (A) Ni^2+-NTA agarose column. A cellular extract from HeLa cells transfected with CCR4^His-FLAG and BTG2^FLAG expression constructs was bound with Ni²⁺NTA agarose. After intensive washes, bound proteins were eluted and analyzed by western blotting using anti-Flag and anti-hCAF1 antibodies. Molecular size markers are given in kilodaltons. (B) Mammalian two-hybrid assay. HeLa cells were seeded at 10⁴ cells/well in 96-well microtiter plates, then transfected after 8 hours using Exgen 500. The transfected DNA included 100 ng of pG4-TK-Luc reporter plasmid together with 50 ng of GAL4 and/or VP16 fusion vectors in the presence or absence of 50 ng of pSG5FlagCAF1, pSG5FlagPOP2 or pSG5Foll. In all experiments, luciferase activity was normalized with the renilla luciferase activity expressed by the pTK-RL vector. Reporter activity was expressed as a ratio of fold induction to the activity of the reporter vector alone.