Fig. 2: Assessment of cross-linking efficiencies.

a Electrophoretic mobility shift assay (EMSA) with increasing amount of NF1 bound to a DNA oligomer harboring its consensus site, or a mutated version of it. The molar ratios of protein to DNA were 2.1:1 (25 ng), 4.4:1 (50 ng), 8:1 (90 ng), and 11:1 (125 ng). The NF1–DNA complex was separated from free DNA by nondenaturing gel electrophoresis and visualized by SYBR Green staining. b NF1–DNA (5′-biotinylated) complex was irradiated with increasing total energy and constant pulse energy of 7 nJ. Samples were separated by denaturing gel electrophoresis, protein–DNA complexes transferred to a nitrocellulose membrane, and biotinylated DNA visualized by probing with an HRP-coupled streptavidin conjugate. Intensities of the cross-linked protein–DNA bands (x-linked species) were quantified and plotted relative to the most intense band at 700 mJ. c NF1–DNA (5′-biotinylated) complex was cross-linked applying increasing pulse energies, and a constant total energy of 1 J. Cross-linked protein–DNA complexes were detected as in b. Band intensities were plotted relative to the most intense band at a pulse energy of 40 nJ. d NF1 bound to a DNA oligo harboring its consensus site or a mutated version of it was irradiated with increasing total energy and constant pulse energy of 50 nJ: cross-linking depended on a functional protein–DNA interaction. e NF1–DNA complex was cross-linked with a pulse energy of 7 nJ and 350 mJ total energy (XL) or left untreated (Ctrl). Cross-linked samples were further optionally treated with DNase I (DN) or proteinase K (PK) and loaded on a SDS-PAGE followed by western blotting. After detection of His-NF1 using an anti-His antibody, the membrane was stripped and reprobed with an HRP-coupled streptavidin conjugate to detect biotin-labeled DNA. The percentage of cross-linked protein–DNA complexes (x-linked species) was calculated as the intensity of the cross-linked band (dashed rectangle) divided by the sum of intensities of all bands observed in the cross-linked sample. f TBP bound to DNA oligos containing either a wild-type (TATAA) or point-mutated (TGTAA) consensus motif were UV irradiated (pulse energy 50 nJ, total energy 1.25 J) and biotin-DNA detected by western blot. Full-scale versions of all blots are depicted in Supplementary Fig. 1.