Pierce cl xposure film

Total protein from cultures of rat SCG neurons was extracted using Cell Lytic (Sigma-Aldrich) and sample buffer from neurons plated on plastic 35-mm dishes coated with 1 mg/ml PDL. The Bio-Rad Mini-PROTEAN Tetra Cell system (Hercules, CA) was used for electrophoretic separation of the protein samples according to the manufacturer's instructions. Protein samples were resolved using 7.5% SDS–PAGE. The gel was transferred onto nitrocellulose membranes (Bio-Rad). The blots were blocked with 5% milk in Tris-buffered saline with Tween 20 (TBST; Bio-Rad), and then probed with primary antibodies at +4°C overnight. After being washed, the blots were incubated with peroxidase-conjugated secondary immunoglobulin G (Jackson Immunoresearch). Membrane-bound peroxidase was visualized on Pierce CL-XPosure Film after treatment with ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA). Levels of protein of interest were normalized with GAPDH for each group and expressed as densitometric ratios against the control lane using ImageJ (Bethesda, MD).