Sc 25392

Cells were lysed using RIPA Buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1% SDS, and 50 mM Tris pH 8.0) containing protease inhibitor cocktail (Pierce Biotechnology, Rockford IL). The lysates were chilled on ice and agitated by vortex every ten minutes for one hour. Total protein concentration was measured by Bradford assay (Bio-Rad laboratories, Hercules, CA). A total amount of 10–40 μg protein per sample was resolved by electrophoresis on a 8% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore Corporation, Bedford MA). Membranes were probed with primary polyclonal rabbit anti-PR antibody (sc-539, Santa Cruz biotechnologies, CA), polyclonal rabbit anti-ERα antibody (sc-543, Santa Cruz Biotechnologies, CA), mouse monoclonal anti-GAPDH antibody (sc-4472, Santa Cruz Biotechnologies, CA), rabbit polyclonal anti-HES1 (sc-25392, Santa Cruz Biotechnologies, CA) or mouse monoclonal anti-ELF-5(sc-376737, Santa Cruz Biotechnologies, CA). The blots were then probed with appropriate horseradish peroxidase conjugated secondary antibody (Vector Laboratories, MD). The protein bands were visualized using enhanced chemiluminescence reagent Hyglo Quick spray (Denville Scientific, South Plainfield, NJ) per the manufacturer's suggested protocol. Relative protein expression was determined by ImageJ (National Institutes of Health, USA).

For Western blotting, 10⁴ cells/µl were used per lane. Immunodetection was performed using 5%-powdered milk in PBS-T (1xPBS, pH 7.4 and 1% Tween20) for blocking (Roth, Germany). Primary antibodies, anti-p21 mouse antibody (OP64; Calbiochem; dilution 1∶200), anti-p16 mouse antibody (550834; BD Pharmingen; 1∶200), anti-p27 rabbit antibody (sc-528; Santa Cruz; 1∶200), anti-γH2AX (07-164; Millipore; 1∶50), anti-Cyclin D1 rabbit antibody (ab16663; Abcam; 1∶500), anti-Cyclin D2 mouse antibody (ab3805; Abcam; 1∶500), anti-Ki-67 mouse antibody (ab6526; Abcam; 1∶200), anti-HES1 rabbit antibody (sc-25392; Santa Cruz; 1∶200), anti-Bcl-2 (IMG-80093; IMGENEX; 1∶200), anti-Bax (IMG-80165; 1∶250) and anti-tubulin mouse antibody (T-9026; SIGMA-Aldrich; 1∶5000) were diluted as indicated in 5%-powdered milk (in PBS-T) and incubated for one hour at room temperature. Washing steps were performed 3×10 min in 1×PBS-T. The secondary horseradish peroxidase-labeled antibodies (Jackson Immuno Research Lab) were incubated for 1 hr at room temperature. Detection of horseradish peroxidase was performed using ECL-detection system and radiographic film (GE Healthcare, Germany). After film development, quantification of signal intensities of the bands in the Western blots was carried out using Metamorph software.

Tissue microarray construction and immunohistochemistry protocol were previously reported [43 (link)]. In brief, primary antibodies against human Jagged1 (ab7771, Abcam; dilution 1:300), ICN1 (ab8925, Abcam; dilution 1:400) and Hes1 (sc-25392, Santa Cruz; dilution 1:300) were applied. A semi-quantitative score was calculated for each case by multiplying the staining intensity (0, negative staining; 1, weak; 2, moderate; and 3, strong) and the percentage of stained cells (0%-100%) at each intensity level. Two experienced pathologists of urology (C. Zhai and Q. Fu) evaluated all staining blinded to the information of patients and the mean value of immunohistochemistry scores were adapted to avoid the inter-observer variability.