ATRX BioID system and proximal associations.
A: Western blot exemplifying stable integration and expression of FLAG-tagged BirA* protein fusions in inducible HEK293 Flp-In T-REx cells (top), and similar expression levels of N- and C-terminally-tagged ATRX (bottom). B: Western blot (top) and Coomassie-staining (bottom) of biotinylated proteins captured on streptavidin beads. C: Example of immunofluorescent labeling demonstrating nuclear targeting of the ATRX-BirA* fusion constructs. D: Gene ontology analysis of ATRX-associating proteins. E: Western blot showing the subcellular distribution of ATRX-associating proteins in HeLa S3 cells. F: Examples of the proximity ligation assay (PLA) showing associations between endogenous proteins in MO3.13 cells. G-H: PLAs showing associations between endogenous ATRX and exogenous FLAG-tagged CCDC71 (G) or FAM207A (H) in HEK293 Flp-In T-REx cells. I: PLAs showing an association between endogenous ATRX and myc-tagged SLF2. Data plots account for ∼100 nuclei in three independent experiments, with the total number of nuclei assessed in brackets. The p-values were obtained using a 2-sided Student’s t-test with unequal variance. J: In vitro interaction between recombinant FAM207A and ATRX.