Inhibition of PRC1 Activity Assay

Inhibition of PRC1 activity was performed to analyse the PRC-dependent and independent activities of RYBP in promoter assays. Sixteen hours after transfection of the required plasmids by CaPO₄ method (detailed in §4.3), HEK293 cells were fed with growth media supplemented with 50 µM of PRC1 inhibitor, PRT4165 (PRT4165, Sigma, cat. no. NSC600157) as previously reported by Ismail et al. and Gracheva et al. [23 (link),24 (link)]. The cells were maintained with PRT4165 supplemented media for further 24 h after transfection and the cells were harvested for whole cell lysates. The cell lysates were then prepared for luciferase reporter assay as described in §4.4.