6708 Nucleic Acids Research, 2007, Vol. 35, No. 20 Figure 5. The pre-RC-binding site is conﬁned to a 119 bp region of fragment B. (A) EMSAs analyses with deleted fragment B (3. 100, 5. 100, 3. 200, 5. 200; see Figure 1A) and wild-type or mutant 119 bp fragments (119, 119.S1–S4). Sequences of the mutant constructs are given in Table 1. (B) Migration of protein-free 119 bp DNA fragments, WT and S1 to S4, in a 4% PAA gel. Table 1. Sequences of the mutated 119 bp fragments 119 (original sequence) 119.S1 119.S2 119.S3 119.S4 50-ACGATGGGGACCGTTTGTGCCACTCGGGAGAAGTGGTGGGTGG GTACGCTGCTCCGTCGTGCGTGCGTGA GTGCCGGAACCTGAGCTCGGGAGACCCTCCGGAGAGACAGAATGAGTGA_30 50-ACGATGGGGACCGTTTGTGCCAAGATGGAGAAGTGGTGGGTGG GTACGCTGCTCCGTCGTGCGTGCGTGA GTGCCGGAACCTGAGCTC GGGAGACCCTCCGGAGAGACAGAATGAGTGA-30 50-ACGATGGGGACCGTTTGTGCCAAGATTTCTAAGTGGTGGGTGG GTACGCTGCTCCGTCGTGCTGCGTGAG TGCCGGAACCTGAGCTC GGGAGACCCTCCGGAGAGACAGAATGAGTGA-30 50-ACGATGGGGACCGTTTGTGCCAAGATTTCTAAGTGGTGGGTGG GTACGCTGCTCCGTCGTGCGTGCGTGA GTGCCGGAACCTGAGAGA TGGAGACCCTCCGGAGAGACAGAATGAGTGA-30 50-ACGATGGGGACCGTTTGTGCCAAGATTTCTAAGTGGTGGGTGG GTACGCTGCTCCGTCGTGCGTGCGTGAGTGCCGGAACCTGAGAGAT TTCTACCCTCCGGAGAGACAGAATGA GTGA-30 Conspicuous sequence elements are depicted in bold letters, introduced mutations are underlined. of DNA replication in myotubes could further be veriﬁed by BrdU incorporation. After ﬁve days in diﬀerentiation medium only two persisting myoblasts were replicating in the examined area (lower panel). Thus, a system of terminally diﬀerentiated cells was available to investigate the expression of pre-RC compo- nents during diﬀerentiation and their interaction with the OBR region in terminally diﬀerentiated myotubes. Expression of pre-RC proteins during myogenic differentiation of C2C12 cells Immunoblot analyses were performed with nuclear and soluble extracts, respectively, prepared from proliferating, conﬂuent or diﬀerentiating C2C12 cells. Subsequently immunoblot analyses of pre-RC components were carried out using speciﬁc antibodies raised against ORC1 and ORC2 (Figure 6B). Sixty hours post-diﬀerentiation, the amount of ORC1 in the nucleus was at undetectable levels. In contrast, soluble ORC1 levels exhibited a peak at 84 h and were then reduced. ORC2 expression remained unaﬀected until 60 h, and even after 132 h ORC2 was detectable at low levels in the nucleus. A soluble variant of ORC2 migrating slower than nuclear ORC2 was observed during diﬀerentiation, probably due to phosphorylation. Immunoblot analysis of proliferating cell nuclear antigen (PCNA) demonstrated nuclear localization exclusively in proliferating myoblasts, not however, in diﬀerentiated myotubes. A slow reduction of soluble PCNA with complete disappearance after 132 h in DM was observed. In summary, protein levels of ORC1 and ORC2 signiﬁcantly decreased in murine C2C12 myotubes during diﬀerentiation. ORC1 could hardly be detected in