Fig. 5.
Fig. 5. EMSAs using the BRE-G1 sequence of the γ-globin gene promoter or TFIID as probe. (A) Nuclear extracts from uninduced K562 or a patient’s erythroblasts (patient no. 5) without treatment are shown by the −; butyrate-induced K562 cells or patient’s erythroblasts on treatment (patient no. 5) are designated by the +. Nuclear extracts prepared from erythroblasts of a normal subject are designated NS. Lane designations are as follows: lane 1, K562 (untreated); lane 2, K562 (butyrate-treated); lane 3, patient no. 5 (before treatment); lane 4, patient no. 5 (on butyrate treatment); lane 5, patient no. 5 (28 hours after treatment); lane 6, patient no. 5 (44 hours after treatment); lane 7, K562 (untreated); lane 8, patient no. 5 (on treatment); lane 9, normal subject (N.S.). Four major protein-DNA complexes, designated a through d, were detected. Arrows denote protein-DNA complexes that appeared in K562 extracts during butyrate treatment. The asterisk denotes a band that is diminished with exposure to butyrate. Nuclear extracts from patient no. 5 are shown before (−), during (+), and 28 and 44 hours after therapy (−28hr and −44hr) are shown. Three new shifted bands are demonstrated during butyrate therapy and persist for at least 44 hours, at which time globin protein synthesis is still altered with an increase in γ-globin and decrease in β-globin compared with baseline. (B) EMSA using a TFIID probe. The origins of nuclear extracts and addition of butyrate are shown on the top of figure. Lane designations are as follows: lane 1, patient no. 5 (before treatment); lane 2, patient no. 5 (on treatment); lane 3, patient no. 5 (28 hours after treatment); lane 4, patient no. 5 (44 hours after treatment); lane 5, K562 (untreated); lane 6, patient no. 5 (on treatment); lane 7, normal subject (N.S.). The arrow indicates the major protein-DNA complex observed with the probe. No new or shifted complexes are observed with butyrate treatment. (C) The wild-type γ-globin promoter probe (W) and a mutant probe (M) were used in EMSA. Probes and the cellular origin of nuclear extracts are shown above the lanes. The positions of the four major protein-DNA complexes, a through d, are shown on the right of figure. A decrease in binding of bands b and c was observed with the BRE-G1 sequence in which mutations were introduced, compared with binding of the normal sequence. (D) Competition assays with an CP2 sequence oligonucleotide. Assays were performed in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 100-fold molar excess of a cold competitor. Probes and the cellular origin of the nuclear extracts assayed are shown above the lanes. Binding of the top band was abolished in the presence of excess cold CP2.

EMSAs using the BRE-G1 sequence of the γ-globin gene promoter or TFIID as probe. (A) Nuclear extracts from uninduced K562 or a patient’s erythroblasts (patient no. 5) without treatment are shown by the −; butyrate-induced K562 cells or patient’s erythroblasts on treatment (patient no. 5) are designated by the +. Nuclear extracts prepared from erythroblasts of a normal subject are designated NS. Lane designations are as follows: lane 1, K562 (untreated); lane 2, K562 (butyrate-treated); lane 3, patient no. 5 (before treatment); lane 4, patient no. 5 (on butyrate treatment); lane 5, patient no. 5 (28 hours after treatment); lane 6, patient no. 5 (44 hours after treatment); lane 7, K562 (untreated); lane 8, patient no. 5 (on treatment); lane 9, normal subject (N.S.). Four major protein-DNA complexes, designated a through d, were detected. Arrows denote protein-DNA complexes that appeared in K562 extracts during butyrate treatment. The asterisk denotes a band that is diminished with exposure to butyrate. Nuclear extracts from patient no. 5 are shown before (−), during (+), and 28 and 44 hours after therapy (−28hr and −44hr) are shown. Three new shifted bands are demonstrated during butyrate therapy and persist for at least 44 hours, at which time globin protein synthesis is still altered with an increase in γ-globin and decrease in β-globin compared with baseline. (B) EMSA using a TFIID probe. The origins of nuclear extracts and addition of butyrate are shown on the top of figure. Lane designations are as follows: lane 1, patient no. 5 (before treatment); lane 2, patient no. 5 (on treatment); lane 3, patient no. 5 (28 hours after treatment); lane 4, patient no. 5 (44 hours after treatment); lane 5, K562 (untreated); lane 6, patient no. 5 (on treatment); lane 7, normal subject (N.S.). The arrow indicates the major protein-DNA complex observed with the probe. No new or shifted complexes are observed with butyrate treatment. (C) The wild-type γ-globin promoter probe (W) and a mutant probe (M) were used in EMSA. Probes and the cellular origin of nuclear extracts are shown above the lanes. The positions of the four major protein-DNA complexes, a through d, are shown on the right of figure. A decrease in binding of bands b and c was observed with the BRE-G1 sequence in which mutations were introduced, compared with binding of the normal sequence. (D) Competition assays with an CP2 sequence oligonucleotide. Assays were performed in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 100-fold molar excess of a cold competitor. Probes and the cellular origin of the nuclear extracts assayed are shown above the lanes. Binding of the top band was abolished in the presence of excess cold CP2.

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