Bcl 2

Cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors [15 (link)–16 (link)]. The protein was resolved by SDS/PAGE and blotted on PVDF membranes (Millipore, Bedford, MA, USA)[15 (link)–16 (link)]. The PVDF membranes were incubated with specific primary antibodies at 4°C overnight [15 (link)–16 (link)]. After incubation with HRP-linked secondary antibodies, immunoreactive proteins were visualized using DAB (Beijing Zhongshan Jinqiao biotechnology Co., Ltd.).
Primary antibody against GRα was from Santa Cruz Biotechnology. IκBα, phospho-p65 and cleaved caspase 3 were from Cell Signaling Technology (Beverly, MA, USA). NF-κB p65, Bcl-2, β-actin and HRP-linked secondary antibodies were from Beijing Zhongshan Jinqiao biotechnology Co., Ltd.

Immunohistochemistry of human samples was performed as previously described [12 (link), 18 (link)]. In brief, 10% neutral formalin-fixed, paraffin-embedded tissues were cut into 5 to 6 μm thickness sections. Immunohistochemistry was done according to the manual of SP-9000-D kits (Streptavidin-Biotin) (Beijing Zhongshan Jinqiao biotechnology Co., Ltd., China). The applied primary antibodies consisted of the following: GRα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NF-κB p65 and Bcl-2 (Beijing Zhongshan Jinqiao biotechnology Co., Ltd.). Expression of GRα, NF-κB p65 or Bcl-2 was evaluated based on the percentage of positive cells. The slide with lower percentage of positive cells than or equal to 5% was considered as negative. The slide with higher percentage was considered as positive.
Immunohistochemistry of cultured cells was performed as previously described [15 (link)]. Cells were cultured on cover slides for 2 days. After fixation in 4% paraformaldehyde for 30 minutes, cells were stained using SP-9000-D kits (Beijing Zhongshan Jinqiao biotechnology Co., Ltd.) according to the manual.

NSC34 cells and mouse spinal cords were homogenized in RIPA lysis buffer (Thermo Scientific, Grand Island, NY, USA) and extract was centrifuged for 10 min at 14,000× g in 4 °C centrifuge. Total protein samples (30–100 µg) were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene difluoride (PVDF) membranes by a wet blotting procedure (100 V, 120 min, 4 °C). Blocking buffer (5%) incubation was followed by incubation with primary antibodies at 4 °C overnight using the following concentration: SOD1 (ab52950; Abcam, Cambridge, MA, USA) 1:1000, Homer1b/c (sc-25271, Santa Cruz, Dallas, TX, USA) 1:1000, Bcl-2 (Zhongshan Jinqiao Biotechnology, Beijing, China) 1:1000, Bax (Zhongshan Jinqiao Biotechnology) 1:1000, and β-actin (Santa cruz Biotechnology, Dallas, TX, USA) 1:1000, which acted as the internal control for normalization of protein expression. Primary antibody incubation was followed by alkaline phosphatase-conjugated secondary antibody. Visualization of bound antibody was achieved with film (Kodak, Rochester, NY, USA) by using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) (Beyotime Institute of Biotechnology, Jiangsu, China).