Ab17851

FFPE tissue sections were dewaxed with xylene and rehydrated in a graded ethanol series before undergoing microwave antigen retrieval in sodium citrate buffer, pH 6.0. Tissue sections were blocked with peroxidase blocking agent (Dako, Cambridgeshire, UK) and then incubated with an anti‐MYB antibody, ab17851 (Abcam, SPM 175). Tissue sections were thereafter washed in phosphate‐buffered saline (PBS) and probed with secondary HRP‐conjugated antibodies, and staining was visualized with 3,3′‐diaminobenzidine (DAB). Haematoxylin was used as a nuclear counterstain. The samples were assessed for MYB positivity; if > 15% of cells demonstrated increased expression of MYB, they were considered positive.

ChIP was performed using the EpiTect ChIP OneDay Kit (#334471, Qiagen, Hilden) according to the instructions of the manufacturer. In brief, 1.5x10⁶ BV-173 cells per sample were lysed and sonicated (5 times 6 s, 0.5 W) using the Vibra-Cell™ system (Sonics, Newtown, USA). Immunoprecipitation was performed with an anti-c-MYB antibody (ab17851, Abcam, Cambridge, UK) and an unspecific control IgG1 control antibody (ab91353, Abcam) according to the manual. For quantification of the immunoprecipitated DNA target, the ChIP qPCR Primer Assay GPH1003144(+)02A (Sabioscience/Qiagen) was applied employing the Roche LightCycler 480 System, using LC480 DNA Master SYBR Green and the standard LightCycler protocol as already described (Roche Diagnostics, Mannheim, Germany). Results derived from triplicate experiments were expressed as percentage of input sample taken before immunoprecipitation during the ChIP procedure. Therefore, the Ct values of the IP fractions were normalized to the input fraction.