Anti syp

Cancer cells were collected and lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentration was measured by BCA assay. Samples in SDS sample buffer were heat-denatured at 95 °C for 5 min. The proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with primary antibodies including anti-MCM2 (sc-373702, 1:1000 dilution), anti-MCM3 (sc-390480, 1:1000 dilution), anti-MCM4 (sc-28317, 1:1000 dilution), anti-MCM6 (sc-393618, 1:1000 dilution), anti-AR (sc-7305, 1:1000 dilution), anti-SYP (sc-17750, 1:2000 dilution), anti-CD56 (sc-7326, 1:500 dilution), anti-cleaved PARP1 (sc-56196, 1:1000 dilution), anti-β-Actin (sc-47778, 1:2000 dilution), and anti-GAPDH (sc-47724, 1:2000 dilution) purchased from Santa Cruz Biotechnology. Secondary HRP conjugated antibody (PI31432, 1:6000 dilution) was purchased from Fisher Scientific. Western blot development and detection was performed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and IVIS Lumina optical imaging system (PerkinElmer). All the raw images of Western blot were summarized in Supplementary Fig. 4.

RIPA (radio immunoassay precipitation) extracts from mouse brain homogenates were used for Western blot analyses as previously described (Di Meco et al., 2014; Giannopoulos et al., 2013). Briefly, samples were electrophoresed on 10% Bis–Tris gels or 3%–8% Tris–acetate gel (Bio‐Rad, Richmond, CA, USA), transferred onto nitrocellulose membranes (Bio‐Rad), and then incubated overnight at 4°C with the appropriate primary antibodies; anti‐5LO [dilution: 1:200] (Santa Cruz, Dallas, TX, USA), anti‐HT7 [1:200] (Thermo, Waltham, MA, USA), anti‐AT8 [1:100] (Thermo), anti‐AT180 [1:200] (Thermo); anti‐AT270 [1:200] (Thermo), anti‐PHF1 (generous gift of Dr. Peter Davies); anti‐PHF13 [1:100 (Thermo)], anti‐SYP [1:300] (Santa Cruz), anti‐PSD95 [1:200] (Thermo), anti‐MAP2 [1:1,000] (Millipore), anti‐GSK3α/β [1:100] (Cell Signaling, Danvers, MA, USA), anti‐pGSK3α/β [1:100] (Cell Signaling), anti‐cdk5 [1:200] (Santa Cruz), anti‐p35/p25 [1:100] (Santa Cruz), anti‐GFAP (Santa Cruz), anti‐CD45 [1:100] (Thermo) and anti‐Beta actin [1:500] (Santa Cruz). After three washings with T‐TBS (pH7.4), membranes were incubated with IRDye 800CW‐labeled secondary antibodies (LI‐COR Bioscience, Lincoln, NE, USA) at room temperature for 1 hr. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). β‐Actin was always used as internal loading control.