PMC:1359074 / 14273-16839 JSONTXT

{"project":"sentences","denotations":[{"id":"T96","span":{"begin":0,"end":95},"obj":"Sentence"},{"id":"T97","span":{"begin":96,"end":172},"obj":"Sentence"},{"id":"T98","span":{"begin":173,"end":318},"obj":"Sentence"},{"id":"T99","span":{"begin":319,"end":447},"obj":"Sentence"},{"id":"T100","span":{"begin":448,"end":480},"obj":"Sentence"},{"id":"T101","span":{"begin":481,"end":653},"obj":"Sentence"},{"id":"T102","span":{"begin":654,"end":1039},"obj":"Sentence"},{"id":"T103","span":{"begin":1040,"end":1069},"obj":"Sentence"},{"id":"T104","span":{"begin":1070,"end":1150},"obj":"Sentence"},{"id":"T105","span":{"begin":1151,"end":1370},"obj":"Sentence"},{"id":"T106","span":{"begin":1371,"end":1435},"obj":"Sentence"},{"id":"T107","span":{"begin":1436,"end":1657},"obj":"Sentence"},{"id":"T108","span":{"begin":1658,"end":1751},"obj":"Sentence"},{"id":"T109","span":{"begin":1752,"end":1856},"obj":"Sentence"},{"id":"T110","span":{"begin":1857,"end":1927},"obj":"Sentence"},{"id":"T111","span":{"begin":1928,"end":2060},"obj":"Sentence"},{"id":"T112","span":{"begin":2061,"end":2140},"obj":"Sentence"},{"id":"T113","span":{"begin":2141,"end":2307},"obj":"Sentence"},{"id":"T114","span":{"begin":2308,"end":2396},"obj":"Sentence"},{"id":"T115","span":{"begin":2397,"end":2467},"obj":"Sentence"},{"id":"T116","span":{"begin":2468,"end":2566},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"text":"Figure 3 Analysis of the Minimal Region (36 bp) of the Proximal ɛy Promoter Responsive to Sox6\n(A) The sequence of the 36-bp fragment and its mutant versions used in EMSA. The WT 36-bp DNA sequence (−63 to −28) of the ɛy globin proximal promoter contains two Sox/Sox6 consensus binding sites, shown in bold underline. Versions with truncation of this sequence (M1) or mutation of one of the two consensus binding sites (M2 and M3) are also shown.\n(B) EMSA with c-Myc-tagged Sox6. EMSA was performed using the 36-bp radio-labeled WT probe (as shown in (A)) and c-Myc tagged Sox6 translated in vitro using reticulocyte lysate (see Materials and Methods). Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, c-Myc antibody (producing a supershift); Lane 5: no competition, Sox6 antibody (producing a supershift); Lane 6: no competion, no antibody using in vitro translated vector containing c-Myc, but not Sox6.\n(C) EMSA with HA-tagged Sox6. EMSA was performed similarly as in (B) using HA-tagged Sox6 translated in vitro. Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, HA antibody (producing a supershift).\n(D) EMSA using MEL cell nuclear extracts and the 36-bp WT probe. Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, Sox6 antibody (producing a supershift).\n(E) EMSA with c-Myc-tagged Sox6, WT and mutant versions of the 36-bp fragment in competition. EMSA was performed using the radio-labeled 36-bp WT probe and the c-Myc tagged Sox6 translated in vitro. Lane 1: radio-labeled free probe; Lane 2: no competition, no antibody. Competition was performed using 200-fold excess cold probes corresponding to WT (Lane 3), M1 (Lane 4), M2 (Lane 5), and M3 (Lane 6).\n(F) Both consensus Sox/Sox6 binding sites are required for Sox6 responsiveness. GM979 cells were transfected with a reporter construct (Figure 2A) containing −63 to +45 of the ɛy proximal promoter together with the CMV-Sox6 overexpression vector. Mutations of the consensus binding sites were also tested (M3, M2, M2 plus M3, see (A)). The fold repression of Sox6 with the WT or mutant constructs is shown. The baseline activities of the mutagenized reporter constructs are comparable to the WT construct."}