Chemiluminescent detection system

To confirm the effect of IL-4 on proliferation of NSCs and to investigate its the mechanism, the expression of proliferating cell nuclear antigen (PCNA) using western blot analysis. Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 140 mM NaCl, 1% (w/v) Nonidet P-40, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 10 μg/ml aprotinin. Cell lysates were separated by 12% SDSpolyacrylamide gel electrophoresis, and electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated with blocking buffer (1× Tris-buffered saline, 1% BSA, 1% nonfat dry milk) for 1 h and then incubated with the primary antibody against PCNA (Santa Cruz Biotechnology; 1:1,000) for overnight at 4°C. Primary antibodies were visualized with peroxidase-conjugated anti-rabbit IgG and a chemiluminescent detection system (Santa Cruz Biotechnology, Inc., USA). The ChemicDoc XRS system (Bio-Rad) was used for visualization and quantification using Quantity One imaging software (Bio-Rad).

For western blots, cell lysates were prepared in RIPA buffer (50 mM Tris-Cl (pH 8.0), 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) with a protease inhibitor cocktail (Roche, Reutlingen, Germany). Total protein concentrations were measured using a BCA kit, and immunoblotting of the clarified cell extracts was performed using 12% SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose membranes (Millipore, Darmstadt, Germany) and incubated at 4 °C overnight with the Cav-1 antibody (BD). An HRP-conjugated anti-rabbit secondary antibody was used for detection with a chemiluminescent detection system (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 140 mM NaCl, 1% (w/v) Nonidet P-40, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF and 10 µg/mL aprotinin. Cell lysates were separated by 8 or 12% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane (Millipore). For the detection of Bax, Bcl-2 and cytochrome c, cells were fractionated into cytosol and mitochondria using a Mitochondria Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The membranes were soaked in blocking buffer (1× Tris-buffered saline, 0.1% Tween 20, 5% nonfat dry milk) for 1 h and incubated overnight at 4°C with the primary antibody. Blots were developed using a peroxidase-conjugated anti-rabbit and anti-mouse IgG and a chemiluminescent detection system (Santa Cruz Biotechnology). The bands were visualized using a ChemiDoc XRS system (Bio-Rad) and quantified using Quantity One imaging software (Bio-Rad). The p-PDK1 (Ser241) and p-Akt (Thr 308 and Ser473) band intensities were normalized to PDK1 and Akt band intensities, respectively. The intensities of cleaved PARP were by the β-actin band intensity, the intensities of Bax were by COX IV intensities and the intensities of Bcl-2 and cytochrome c were adjusted by the intensities of α-tubulin intensities.