Western blot analysis of c-Fos expression in rat C6 cells (40 ug lysate loading per lane). C6 cells were serum-starved for 20 hours and then stimulated with 20% FBS for 2 hrs (+). Control cells were left in serum-depleted culture medium (-). M-1752-100 and R-1751-50 detect a strong band at 50 kDa in stimulated cells but not in control cells, representing increased c-FOS expression.
Western Blotting Method: SDS-PAGE: denaturing and reducing, 4-12% Bis-Tris gel; Transfer: Tris-Glycine (Towbin's) buffer with 20% methanol; Membrane: PVDF (0.45 µm); Blocking: 5% skim milk in TBST, 1 hour at RT; Primary antibodies: R-1751-50 (1 µg/mL), M-1752-100 (1 µg/mL), incubation overnight at 4°C; Secondary antibodies: anti-rabbit-HRP (1/6000) or anti-mouse-HRP (1/3000), 1 hour at RT; Detection: Chemiluminiscence.
Left: Rat hippocampus stained for c-FOS (red) by Immunohistochemistry. Section was co-stained with rabbit antibody to FOX3/NeuN (R-3770-100, green). Blue: DAPI nuclear stain. The hippocampal neurons stain green for FOX3/NeuN and a few also are expressing c-FOS, and thus appear orange. These cells were spontaneously active at the time the animal was sacrificed. Right: Western blot analysis of c-FOS expression (green) in cell lysates. Mouse antibody to c-FOS was used at 1:1,000 dilution. GAPDH (red, lanes 2-5) was used as loading control (rabbit antibody to GAPDH, R-1701-100, 1:20,000).  protein standard,  HeLa cells in serum free media,  HeLa cells stimulated with 20% FBS for 2 hours after 36 hours serum starvation,  rat cortical neurons,  rat cortical neurons treated with membrane depolarization buffer for 5 hours. Multiple bands at 50-65 kDa in stimulated or treated cell lysates correspond to different forms of the c-Fos protein. The single band at 37 kDa (red) represents GAPDH protein.