Figure 2

The setup of the two screening assays. (a) Schematic representation of the HTS assay. The LEM-domain of human emerin is immobilized on the bottom of a multiwell plate. In the presence of BAF, the ATTO 425-labelled polymerase chain reaction (PCR) product is retained in the well or washed away if any part in the LEM-BAF-DNA chain is missing. (b) The HTS assay was performed with increasing concentrations of BAF (circles), without the LEM-domain (up triangles) or without DNA (down triangles). Fluorescence was normalized with respect to the BAF (1000 nM) value (BAF + LEM + DNA). Mean ± SD (N = 4). (c) ESI–MS analysis of BAF (10,056 Da) without (black peaks in dashed box) and with preincubation with human VRK1 (red peaks in clear box). (d) The HTS assay was conducted with or without VRK1 pre-treated BAF. Fluorescence was normalized and statistical significance tested with respect to the sample without VRK1 pre-treatment. Mean + SD (N = 3). ***p < 0.01. (e) Schematic representation of the DNA fragment retention (DFR) assay. A 270-bp ATTO 425-labelled and biotinylated PCR product is immobilized on a streptavidin coated multiwell plate. Upon the addition of BAF and a restriction enzyme (here EcoRI), the fluorescently labelled DNA terminus is cleaved off and is only retained in the well due to BAF clustering. (f) The DFR assay was performed with increasing BAF concentrations, both in the presence (circles) or absence (triangles) of EcoRI. Fluorescence was normalized with respect to the BAF (0 nM) value w/o EcoRI. Mean ± SD (N = 3).