Binding of the isolated Tax N-terminus to CDK4 and cyclin D2. A) Physical map of Tax's functional domains and the position of the N-terminal peptide B) Taxwt and TaxM1-R40 were produced in E. coli and coupled to S-protein agarose. The figure depicts a coomassie brilliant blue-stained SDS-PAA gel loaded with the purified protein coupled to S-protein agarose and samples before and after induction with IPTG. C) CDK4, cyclin D2 and cyclin E were translated in vitro and incubated with S-agarose coupled, E.coli-produced Taxwt and TaxM1-R40. Bound proteins were detected in gels by phosphoimaging (precipitation). To control for equal inset, aliquots of the radioactive proteins were subjected to gel electrophoresis (input). D) The radioactive signals of bound proteins of two independent experiments were quantitatively evaluated. The figure depicts the mean relative binding. E) For in vivo pull-down analysis, cyclin D2 and cyclin E plasmids were transfected into 293T cells. Lysates were incubated with S-agarose coupled to Taxwt or the N-terminal peptide (TaxM1-R40). Bound proteins and aliquots of the lysates were subjected to gel electrophoresis and immunoblotting, using polyclonal cyclin D2 and cyclin E antibodies.