Subcellular distribution and DNA-binding activity of BAF mutants.

<p>(A) Immunofluorescence analyses of FLAG-BAF-MTTSQ and FLAG-BAF-MAAAQ with an anti-FLAG antibody (AlexaFluor488-conjugated secondary antibody). (B) Subcellular fractionation analyses of cells indicated in (A). Cell lysates were fractionated to saponin-soluble cytosolic fraction (grey bars) and insoluble nuclear fraction (black bars). Each fraction was analyzed by western blot with an anti-FLAG antibody and quantified with ImageLab software (BioRad) (n = 3). % fraction was calculated from the amount of protein on each fraction relative to the total protein level. Error bars represent standard deviations. (C) ChIP analyses of FLAG-BAF-MTTSQ and BAF-MAAAQ. Cells were transfected with 150 ng of pUC-Neo plasmid DNA for 24 h followed by fixation, immunoprecipitation with anti-FLAG antibody, and reverse crosslinking of protein-DNA complexes. Purified DNA was analyzed by qPCR using primers specific for the pUC-Neo plasmid (Plasmid) or the β-actin locus of chromatin DNA (Genomic). Fold enrichment was obtained relative to empty vector control and normalized to input DNA. Error bars represent standard deviations.