Electrophoretic mobility shift assay (EMSA)
EMSA was performed as previously described 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 the COL13 promoter, located between 676 and 1675 bp
upstream of the transcription start site (ATG) (Fig. 4b). 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 the core-binding
motif of the 1059 bp region, a series of EMSAs involving deletions of
this region were performed. 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 is listed in
Supplementary Table 2.