Fig. 7

In situ cell extraction applied to genetic studies of chromatin binding for candidate drug targets TRIM24, SMARCA2 and ATAD2. a Schematic representation of proteins highlighting the multi-domain nature of select bromodomain-containing genes. b A functional TRIM24 bromdomain is required for chromatin binding. IF images of HeLa cells expressing FLAG-tagged wild-type (WT) or bromodomain-binding-deficient mutant (BRD mut) forms of full-length TRIM24 under in situ cell extraction (+) and non-extraction (−) conditions. c Quantification of the indicated FLAG-tagged full-length proteins (ATAD2, TRIM24 and SMARCA2) in the nuclei of transfected HeLa cells after in situ cell extraction (CSK buffer with 100 mM NaCl) for wild-type (black) and bromodomain mutant (green) proteins (n = 3; similar results observed across different cell lines including HeLa, hMECs and A549; data not shown). d Representative IF images of hMECs expressing FLAG-tagged full-length (WT) and mutant ATAD2 constructs (BRD mut and ATPase dead) followed by IF detection of the ATAD2 transgene (i.e., anti-FLAG antibody). e Quantification of the nuclear signals for ATAD2 wild-type (WT), BRD mutant and ATPase dead proteins after in situ cell extraction using increasing NaCl stringency. Changing the stringency of the in situ cell extraction (0–500 mM NaCl) does not change the relative rank order of their chromatin-binding affinities