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. 2019 Mar 20;61(3):228–251. doi: 10.1111/dgd.12601

Figure 4.

Figure 4

HOXA11 and HOXA13 bind to multiple HBSs in the CHBRL in vitro. EMSAs using 741b, a part of the Shox2 CHBRL/m741 enhancer (VISTA), as a probe. (a) Multiple shift bands emerged in a HOX protein dose‐responsive manner; (−): no protein; MBP: 153 ng of MBP was added to the reaction. The amount of MBP‐HOXA13HD used was 7.6, 15.2, 42, 85, 152, 152, 306 and 612 ng. (b) Band shift was competed by HBS‐containing oligonucleotides HBS1 (TTAT) and HBS2 (TAAT) but not by their mutant forms (T to G and A to C). 153 ng of MBP and 128 or 255 ng of MBP‐HOXA13HD were used. 5‐fold or 50‐fold excess molar unlabeled competitor was added to the reaction. (c) Multiple shift bands emerged in a HOX11/13 protein dose‐responsive manner; (−): no protein, MBP; 153 ng of MBP was added to the reaction. The amount of MBP‐HOXA11HD added was 1.4, 2.8, 5.7, 11.4, 28, 57, 57, 114, 228 ng. (d) Band shift was competed by HBS containing oligonucleotides HBS1 (TTAT) and HBS2 (TAAT) but not by their mutant forms (T to G and A to C). 153 ng of MBP and 95 or 189 ng of MBP‐HOXA11HD were used. 5‐fold or 50‐fold molar excess unlabeled competitor was added to the reaction. (e–h) Multiple shift bands emerged in the HOX11/13 protein in a dose‐dependent manner and were specifically competed by HBS‐containing oligonucleotides. (e, g) MBP‐HOXA13HD and (f, h) MBP‐HOXA11HD. (e) and (f) Aff3 probe, (g) and (h) Bmp2 probe. The sequence of the probe and conserved HBS are shown in Supporting Information Figure S5I, J. Arrowhead and arrow indicate the shift band and free probe, respectively. C: competitor, P: protein.