Mouse anti gapdh

Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Roswell, GA, USA) on ice for 30 min; samples were then centrifuged at 12 000 × g at 4 °C for 20 min to collect the supernatants. Protein concentrations were measured using the BCA Protein Assay Kit (Thermo Scientific, USA). Samples of 50 μg were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies, including rabbit anti-PTCH1 (1:1 000, Abcam, Cambridge, UK; catalogue No.: ab53715, detecting the N terminal of human PTCH1), anti-GLI1 (1:1 000, Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:5 000, ZSGB-BIO, Beijing, China), respectively. After three washes for 5 min each in TBST, membranes were incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA; diluted at 1:10 000) at room temperature for 1 h. Finally, the protein bands were visualized using the enhanced chemiluminescence method and an ECL detection kit (CWbiotech, Beijing, China). Band densities were analyzed by Gel-Pro Analyzer 4.0 software, and protein levels were normalized to those of GAPDH.

Cells were lysed with cell lysis buffer (CST, MA, USA; Cat #9803) in the presence of protease inhibitors. 40 μg of total protein were electrophoresed on 12% SDS-PAGE and electrophoretically transferred onto a PVDF membrane, blocked with 5% skim milk at room temperature (RT) for one hour. Membranes were later probed with different primary antibodies overnight at 4 °C. The membranes were washed for 5 min three times in TBS with 0.1% Tween-20 and then incubated with horseradish peroxidase-conjugated mouse (Cat #1706516, Biorad, CA, USA) or rabbit (Cat #1706515, Biorad, CA, USA) secondary antibodies at RT for one hour. The membranes were washed three times for 5 min in TBS with 0.1% Tween-20, and then visualized with the Lumi-Light Western Blotting Substrate (Roche, Basel, Switzerland; Cat #12015200001) on the 5200 chemiluminescence imager (Tanon, Shanghai, China). The following primary antibodies were purchased: mouse anti-PTGES (Santa Cruz, CA, USA; Cat #sc-166,309), rabbit anti-Cleaved Caspase-3 (CST, MA, USA; Cat #9661), mouse anti-Cleaved PARP-1 (Santa Cruz, CA, USA; Cat #sc-56,196), mouse anti-GAPDH (ZSGB-BIO, Beijing, China; Cat #TA-08).

The hippocampal samples were separated using 10% SDS-PAGE and transferred to PVDF membranes. Then, membranes were probed with mouse anti-CaMKII-α and II-β (1 : 1000, CST), rabbit anti-GFAP (1 : 1000, CST), rabbit anti-F-actin (1 : 1000, CST), and mouse anti-GAPDH (1 : 10000, ZSGB-BIO) at 4°C overnight. After washed with TBST, the membranes were incubated with goat anti-rabbit (1 : 3000, CST) for 1 h at room temperature. Blots were developed using a chemiluminescent substrate and analyzed with the Quantity One software (version 4.4).