Phospho stat1

Four cell lines were all purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), including human neuroblastoma BE(2 (link))-C and SH-SY5Y, rat Schwann cell (RSC) RT4-D6P2T, and human Schwann cell (HSC) from ScienCell Research Lab (San Diego, CA, USA). Following the manual, the cell culture medium and related reagents were used, and all were purchased from the cell culture facility at University of California, San Francisco. Recombinant human BDNF was purchased from R&D systems Inc. (Minneapolis, MN, USA) and used for the treatment. S100, p75 antibodies (Chemicon, Inc., Temecula, CA, USA) and secondary antibody FITC-conjugated goat anti-mouse IgG (Chemicon, Inc., Temecula, CA, USA) were used for immunofluorescence staining. Beta-actin, JAK2, phospho-JAK2 (Chemicon, Inc., Temecula, CA, USA), STAT1, phospho-STAT1, STAT3, and phospho-STAT3 (BD Biosciences, CA, USA) antibodies were used for immunoblot. The reagents in ECL kit (Amersham Life Sciences Inc., Arlington Heights, IL, USA) were used as substrate for all the immunoblotting. OSM-M and IL-6 enzyme linked immunosorbent assay (ELISA) kit (R&D systems Inc., Minneapolis, MN, USA) were applied to assay the secretion of cytokine from Schwann cells.

Cells were harvested and lysed with 1× RIPA (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris [pH 8.0]) with phosphatase and protease inhibitors (Sigma-Aldrich) for 5 min at 37°C. Lysates were collected, and equal protein amounts were separated by SDS-PAGE and blotted onto a nitrocellulose membrane by wet blotting (Bio-Rad). Membranes were blocked with TBS-T containing 5% bovine serum albumin (BSA) for 2 h at room temperature. The primary antibodies beta-actin (Sigma #5441) and phospho-STAT1 (BD Transductions, catalog no. 612233) were diluted in the same blocking buffer, followed by incubation overnight at 4°C. Membranes were then washed three times with TBS-T for 10 min at room temperature with rocking. Anti-mouse antibodies coupled with horseradish peroxidase (HRP; GE Healthcare, catalog no. NA934V) were used at a 1:5,000 dilution in blocking buffer, followed by incubation at room temperature for 1 h with rocking. Membranes were washed three times with TBS-T for 10 min at room temperature with rocking. HRP detection reagent (GE Healthcare) was mixed 1:1, followed by incubation at room temperature for 5 min. Membranes were exposed to film and developed.