In vivo S -nitrosylation of actin in the spinal cord of inflammatory pain model. A. Time course of S-nitrosylation of proteins in the spinal cord. The dorsal spinal cords at the L3-L5 levels were dissected at 0, 5, 30 or 60 min after injection of 2% formalin (5 μl) into the hindpaws, and homogenates prepared. The soluble fraction of the homogenates was subjected to the biotin-switch assay. S-Nitrosylated and total actin were detected by anti-biotin and anti-actin antibodies, respectively, as described under "Methods." An arrow indicates the position of S-nitrosylated actin (S-NO-actin). B. Purification of S-nitrosylated actin on streptavidin-agarose. After the soluble fraction prepared from the dorsal spinal cord at 5 min after formalin injection was subjected to the biotin-switch method, S-nitrosylated proteins were purified on streptavidin-agarose. The eluate was resolved on 10% SDS-PAGE, and S-nitrosylated actin was detected with anti-actin antibody. Authentic actin (1 μg) was used as a positive control.