Electrophoretic mobility shift assay on native agarose gel. Free importin
α5 (24 μg), pSTAT1 (25 μg) and the pSTAT1:α5
complex (60 μg) are in lanes 1, 2 and 3, respectively. Addition of
0.5-, 1.0-, 2.0-fold molar excess (over importin α5) of a 38-mer
dsDNA oligonucleotide containing two
cfosM67-promoter elements to 60 μg of
pSTAT1:α5 displaces importin α5 from pSTAT1 and yields a new
pSTAT1:DNA complex (in lanes 4, 5, 6, respectively). A control of the
pSTAT1:DNA complex formed by adding an excess of DNA to 25 μg pSTAT1
is in lane 7. (B) Importin α5 titration on native
agarose gel. 4, 8, 12, 16, 20, and 24 μg of importin α5 were
loaded in lanes 1-6, respectively. (C) Standard curve
calculated for the intensity of importin α5 bands in panel B. The
band intensity was quantified densitometrically. A line of best fit was
calculated using the ordinary least squares method in KaleidaGraph
®. The error bars represent the standard deviation of three
independent quantifications.