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. Author manuscript; available in PMC: 2018 Sep 27.
Published in final edited form as: Cell. 1991 Jun 28;65(7):1255–1266. doi: 10.1016/0092-8674(91)90020-y

Figure 3. In Vitro DNA Binding of the TRβ Protein to MHC TRE Mutants.

Figure 3.

As a radiolabeled probe for the gel retardation DNA-binding assay, the MHC-N double-stranded oligonucleotides were labeled by a filling-in reaction with Klenow enzyme in the presence of [α−32P]dCTP. TRβ protein was overexpressed in COS cells by transfecting 20 μg of pCMX-TRβ plasmid. As a control, cell extracts were also prepared from mock-transfected (20 μg of pCMX-LUC) COS cells. Protein (5 μg) of the mock- (control) or TRβ-transfected cell extracts was incubated with the MHC-N probe, and the protein-DNA complex was resolved electrophoretically according to Damm et al. (1989). With the TRβ extracts, 50-fold excess amounts of cold oligonucleotides as indicated were added as a competitor for the TRβ binding to the MHC-N.