Spinal Cord Injury Protein Analysis

Whole-cell lysates from 0.5-cm sections of spinal cord tissue containing the injury epicenter, cortical neurons, and PC12 cells were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer. Protein samples (50 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies against TGF-β1 (1:500, Abcam, UK), PLOD2 (1:500, Proteintech, Chicago, IL, USA), P-Smad3 (1:300, Cell Signaling Technology, Boston, USA), Smad2/3 (1:1,000, Cell Signaling Technology, USA), P-AKT (1:5,000, Abcam, UK), AKT (1:10,000, Abcam, UK), NSE (1:2,000, Proteintech), GFAP (1:500, Abcam, UK), GAP43 (1:1,000, Abcam, UK) and GAPDH (1:2,500, Proteintech, USA) at 4° C overnight. The next day, the membranes were incubated with corresponding secondary antibodies (1:10,000 dilution) at room temperature for 1 h [37 (link)]. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) and band density quantified using Image J software.