Electrophoretic mobility shift assay (EMSA)
EMSA was performed as our previously described paper with the LightShift
Chemiluminescent EMSA kit (Thermo Scientific, United States)
(Han et al., 2017). The dual-luciferase
assay mapped the COL3 binding site to a 1059-bp region of theCOL13 promoter, that located between 676 and 1675 bp upstream of
the transcription start site (ATG) (Fig. 5b). This promoter region was
used as a 5’ end biotin labeled probe and the same fragment, but
unlabeled, was used as a competitor. To investigate core-binding motif
of the 1059-bp region, a series of EMSAs involving deletions of this
region were done. We divided the 1059-bp promoter sequence into five
overlapping regions: -1675 to -1391 bp (probe 1), -1421 to -1184 bp
(probe 2), -1201 to -1040 bp (probe 3), -1060 to -868 bp (probe 4), and
-898 to -616 bp (probe 5). The sequence of probes are listed in
Supplementary Table 2.