TI - Constitutive expression of active topoisomerase IIalpha/beta-chimeras in HEK 293 cells . AB - When transfected into HEK 293 cells , each of the constructs depicted in Figure 1B gave rise to viable cell lines supporting stable expression of the YFP-fused proteins . Cell clones with intermediate expression levels were selected and expanded for further analysis . All clones exhibited growth rates and gross morphologies similar to cells not transfected or expressing YFP alone . To assess the integrity of the fusion proteins and to compare their relative expression levels , we subjected the cells to western blotting and probed the blots with YFP antibodies . YFP-fused full-length , non-chimeric topoisomerase IIalpha and IIbeta and the various topoisomerase IIalpha/beta chimeras were detected as single protein bands of expected size . There were no smaller bands detected in addition by YFP antibodies ( Figure 2A , top , odd numbered lanes ) . Thus , we could exclude rearrangements of the transgenes and safely assume that yellow fluorescence of the cells was entirely due to the desired YFP-fused protein . All constructs supported similar expression levels allowing a comparison of data between these cells . To compare YFP-fused and endogenous enzymes , blots were probed with isoform-specific antibodies against C-terminal epitopes of topoisomerase IIalpha or IIbeta ( Figure 2A , middle and bottom , respectively ) . The YFP-fused proteins could clearly be discriminated from the corresponding endogenous enzymes as additional bands of slower migration . From comparison of lanes it became evident that endogenous levels of topoisomerase IIalpha and IIbeta were similar in all transfected cell clones ( Figure 2A , middle and bottom , odd numbered lanes ) and similar to those in untransfected cells ( not shown ) , indicating that none of the YFP-fused enzymes interfered with endogenous topoisomerase II expression . It should also be noted that the desired exchanges of CTRs outlined in Figure 1B were confirmed by the presence of unique C-terminal epitopes of topoisomerase IIalpha and IIbeta in the products of the various constructs shown in middle and bottom panel of Figure 2A . To compare expression levels of endogenous and YFP-fused proteins within each clone , we intended to compare signal intensity within the lanes of western blots stained with isoform-specific topoisomerase II antibodies ( Figure 2A , middle and bottom , odd numbered lanes ) . However , upon testing several antibodies directed at various unique epitopes of human topoisomerase IIbeta , we found that all antibodies tested did not exhibit the same preference for the endogenous form and heterologously expressed YFP-fused variants of the enzyme . On the contrary , the antibodies preferentially recognized one or the other form of the enzyme (Figure 2B) . Thus , conclusions about expression levels drawn from such analyses are in our eyes unreliable . However , it should be noted that all cell clones expressing YFP-fused enzyme constructs exhibited growth rates and morphologies indistinguishable from untransfected cells , suggesting that the enzymes were at least expressed at physiologically tolerable levels . Similar analyses were carried out on cell clones expressing YFP fusion proteins of the CTRs of topoisomerase IIalpha and IIbeta alone ( Figure 2C ) . These constructs also gave rise to single protein bands . However , the apparent molecular weight ( ~100 kDa in both cases ) was larger than expected from the amino acid sequence ( 70 kDa for alpha CTR-YFP and 80 kDa for beta CTR-YFP ) . This could be due to phosphorylaTION , since these regions harbor the majority of PHOSphorylation sites ( 38 ) .