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. Author manuscript; available in PMC: 2016 Mar 15.
Published in final edited form as: Exp Physiol. 2009 Aug 14;94(12):1163–1173. doi: 10.1113/expphysiol.2009.049692

Figure 2. The NF1 binding site specifically interacts with nuclear proteins.

Figure 2

A, serial mutations determine specific binding site. An EMSA using an 80 bp radiolabelled oligonucleotide probe ‘−1967 to −1887’ was performed, and all reactions contained the same sample of nuclear protein extracted from control left ventricular muscles (20 μg). Binding specificity was analysed through competition assays by pre-incubating extracts with 150-fold molar excess of various unlabelled oligonucleotides, as follows: −1967 to −1887 wild-type (self; lane 2); NF1mut (lane 3); RARmut (lane 4); GRmut (lane 5); GATAmut (lane 6); SEFmut (lane 7); and TTFmut (lane 8). Lanes 1 and 9 contain no competitors (probe only). The sample in lane 10 used a 80mer −1967 to −1887 containing a mutated NF1 site as the radioactive probe; it contained no other competitors. The arrow labelled ‘Sp’ indicates the location of the specific band. Underneath the picture of the gel is the sequence of the wild-type probe with the putative transcription factor binding sites underlined and labelled. Abbreviations: GR, glucocortoicoid receptor; RAR, retinoic acid receptor; SEF, soybean embryo factor; and TTF1, thyroid nuclear factor 1. The exact sequences of each oligonucleotide are shown in Table 1. B, intact NF1 binding site is important to formation of specific complex. All reactions contained the same sample of nuclear protein extracted (NE) from control left ventricular muscles (20 μg). With the exception of the sample in lane 1, samples in lanes 2–10 contained an 37 bp radiolabelled oligonucleotide probe, wild-type βAS −1959 to −1922. Lanes 1 and 2 contain no competitors (probe and NE only); lane 1 contains #2 NF1 mutant probe and lane 2 contains wild-type βAS −1959 to −1922. Binding specificity was analysed through competition assays by pre-incubating extracts with 150-fold molar excess of various unlabelled oligonucleotides, as follows: wild-type (self; lane 3); #2 NF1 mutant (lane 4); #3 NF1 mutant (lane 5); #4 NF1 mutant (lane 6); #1 NF1 double mutant (lane 7); #5 NF1 mutant (lane 8); #6 NF1 mutant (lane 9); and RAR mutant (lane 10). The arrows labelled ‘Sp’ indicate the location of the specific bands. The sequences of these mutants are as follows: wild-type, GGTTTTTGTCGCTTGGACCTGACCCAGGCTGACCCAA #1, GGTTTTTGTCGCTaGGACCTGACCaAGGCTGACCCAA #2, GGTTTTTGTCGCTaGGACCTGACCCAGGCTGACCCAA #3, GGTTTTTGTCGCaTGGACCTGACCCAGGCTGACCCAA #4, GGTTTTTGTCGCTTcGACCTGACCCAGGCTGACCCAA #5, GGTTTTTGTCGCTTGGAaCTGACCCAGGCTGACCCAA #6, GGTTTTTGTCGCTTGGtCCTGACCCAGGCTGACCCAA RARmut (80 bp) CAAAGCCT GGTTTTTGTCGCTTGGACCcGtaCCAGGCcGtaCCAATGTTCTCAGTGCCTTATCATGCCTCAAGAGCTTG Putative site sequences span 14 bases (bold), with wild-type being the wild-type NF1 site and #1 to #6 being a series of mutants within the NF1 site (mutated nucleotides are underlined and lowercase). The RAR mutant is 80 bp as used in Fig. 2A. C, NF1 antibody immunodepletes and causes supershifted bands of the specific complex. This EMSA used the same nuclear protein sample and wild-type sequence 37 bp −1959 to −1922, which is the same as the radioactive probe used in B. The lanes are as follows: ‘probe only’ has no competitor or antibody added (lane 1); wild-type −1959 to −1922 (self; lane 2); NF1 antibody (NF1 Ab, 4 μg; NF1c (H-300):sc-5567; lane 3); thyroid hormone receptor TRα antibody ‘215’ (4 μg; Affinity Bioreagents, Golden, CO, USA; lane 4); thyroid hormone receptor TRα antibody ‘216’ (4 μg; Affinity Bioreagents; lane 5); and NF1 antibody 2X (8 μg; NF1c (H-300):sc-5567, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; lane 6). The arrows labelled ‘Sp’ indicate the location of the specific bands. The arrows on right labelled ‘SS’ indicate the supershifted bands at the top of the gel. Dotted arrows indicate bands supershifted using TRα ‘215’ antibody. (Pre-immune serum challenge is not shown.)