A. Western blot screening of three MC3T3-E1-derived lines stably expressing either of two shRNA sequences targeting pRB. The constitutive protein hsc70 is a loading control.
B. Staining at day-6 post-induction shows impaired induction of alkaline phosphatase in the pRBdepleted lines.
C. ChIP analysis in the Seq1.AA1 line. Depletion of pRB results in failure to displace KDM5A, persistence of H3K4 mono-methylation, and failure to recruit BRG1 and RNA polymerase-II.
D. Schematic illustration of the roles of p300 and pRB during the switch from: (i) the repressed state characterized by BRM-specific SWI/SNF occupation and the presence of KDM5A, to: (ii) the activated state characterized by displacement of KDM5A, and recruitment of BRG1-containing SWI/SNF. The unlabeled circles represent the non-enzymatic components of the SWI/SNF complex.