Activation of P38 MAPK and NFκB protein by Western Blotting

Western blotting on cell lysates was performed as described previously [19]. Briefly, microglia and astrocytes were exposed to NaSH and STS at 100 μM for 8 h and were subsequently exposed to stimulants for 2 h. Human microglia and astrocytes were treated with a lysis buffer (150 mm NaCl, 12 mm deoxycholic acid, 0.1 % Nonidet P-40, 0.1 % Triton X-100, and 5 mm Tris-EDTA, pH 7.4). The protein concentration of the cell lysates was then determined using a BCA protein assay reagent kit (Pierce, Rockford, IL). Proteins in each sample were loaded onto gels and separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (150 V, 1.5 h). The loading quantities of lysate proteins were 100 μg. Following SDS-PAGE, proteins were transferred to a PVDF membrane (Bio-Rad, CA) at 30 mA for 2 h. The membranes were blocked with 5 % milk in PBS-T (80 mm Na₂HPO₄, 20 mm NaH₂PO₄, 100 mm NaCl, 0.1 % Tween 20, pH 7.4) for 1 h and incubated overnight at 4 °C with a polyclonal anti-phospho-P38 MAP kinase antibody (9211, Cell Signaling, Beverly, MA, 1/2,000) or anti-phospho-P65 NFκB antibody (3031, Cell Signaling, 1/1,000). The membranes were then treated with a horseradish peroxidase-conjugated anti-IgG (P0448, DAKO, Mississauga, Ontario, CA, 1:2,000) or the secondary antibody anti-mouse IgG (A3682, Sigma, 1/3,000) for 3 h at room temperature, and the bands were visualized with an enhanced chemiluminescence system and exposure to photographic film (Hyperfilm ECL™, Amersham Biosciences). Equalization of protein loading was assessed independently using α-tubulin as the housekeeping protein. The primary antibody was anti-α-tubulin (T6074, Sigma, 1/2,000) and the secondary antibody anti-mouse IgG (A3682, Sigma, 1/3,000). Primary antibody incubation was overnight at 4 °C, and the secondary antibody incubation was for 3 h at room temperature.