The neurospheres were isolated, suspended in RIPA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with a protease inhibitor cocktail (Sigma-Aldrich), triturated and centrifuged at 10 000 g for 10 min at 4 °C. The resultant supernatants were separated on 10% SDS polyacrylamide gel electrophoresis gels (10 μg protein per lane), and the proteins were blotted onto a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk, the membranes were incubated with either anti-p38α antibody (Cell Signaling Technology; dilution 1/1000) or anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1/1000) at 4 °C overnight, followed by incubation with HRP (horse radish peroxidase)-conjugated anti-rabbit IgG (GE Healthcare, Little Chalfont, UK; dilution 1/5000) or HRP-conjugated anti-goat IgG (GE Healthcare; dilution 1/5000) at room temperature, for 1 h. Detection of the signals was carried out with an Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore), and the bands were analyzed using the LAS-1000 plus image analyzer (Fuji Film, Tokyo, Japan). The intensities of the bands were quantified using the Image Gauge software (Fuji Film). The expression levels of p38 were normalized to that of GAPDH.
