Western blot and RNA immunoprecipitation assay was performed as previously described[29 (link)]. Immunohistochemistry (IHC): Phospho-Rb, total Rb, Ki67 and TUNEL staining were performed on 10% paraffin embedded tumor slides per manufacture protocol guidelines and imaged using Leica florescence microscope at 20x and 40x magnifications. IHC staining was performed by our pathology department, Dr. Wei Jiang and Raymond O’Neill, at Thomas Jefferson University.
Western antibodies: Phospho-Rb (1:500, Cell Signaling Technology, Danvers, MA, catalog #9307), total Rb (1:500, Cell Signaling Technology, catalog #9309), HuR (1:1000, Life Technology/ Thermo Fisher, Waltham, MA, catalog# 390600), YAP1 (1A12) (1:500, Cell Signaling Technology, catalog# 12395), cyclin D1 (1:500, Santa Cruz, Santa Cruz, CA, catalog# 8396), p27 (1:1000, Santa Cruz, Santa Cruz, CA, catalog #sc-1641) and alpha-tubulin (1:5000, Invitrogen, Waltham, MA, catalog# A11126) primary antibodies used for protein evaluation by western blot.
Immunohistochemistry (IHC): Phospho-Rb (Cell signaling technology, catalog #8516), total Rb (Cell signaling Technology, catalog #9309) and Ki67 (Roche, Branchburg, NJ, catalog #790–4286) and TUNEL (Trevigen TACS 2 TdT Core Kit, Gaithersburg, MD, catalog# 4810-30-CK) staining antibodies were used for IHC analysis. For TUNEL staining, only diaminobenzidine (DAB) staining was used.