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Figure 1 | Molecular Pain

Figure 1

From: Involvement of S-nitrosylation of actin in inhibition of neurotransmitter release by nitric oxide

Figure 1

Identification of protein S -nitrosylated by SNAP in the spinal cord as actin. A. S2 and P2 fractions of mouse spinal cords were incubated with the indicated concentrations of SNAP for 1 h and subjected to the biotin-switch assay for S-nitrosylation of proteins. Samples (5 μg of each) were resolved by non-reducing SDS-PAGE and immunobloted with anti-biotin antibody. B. The S2 fraction was incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. The biotinylated proteins were purified by using a streptavidin-agarose gel, and the bound proteins were eluted by SDS-sample buffer containing 2-mercaptoethanol. Eluates were analyzed by SDS-PAGE, and proteins were visualized by silver staining. Three major bands were identified as β-tubulin, β- and γ-actin, and glyceraldehyde-3-phosphate dehydorogenase (GAPDH). C. MALDI-TOF MS spectrum used for identification of actin. Twelve matched mass peaks are indicated. D. Amino acid sequence of mouse γ-actin. The parts of the sequence in red correspond to the peptide fragments obtained by tryptic digestion. The amino acid sequence of β-actin completely matches residues 20-351 of γ-actin except that P at the 31st residue of γ-actin was replaced by S at the 12th residue of β-actin. E. S-Nitrosylation of actin by SNAP in the S2 fraction, but not in the P2 fraction, of the spinal cord. S2 and P2 fractions of spinal cords were incubated with 100 μM SNAP for 1 h and subjected to the biotin-switch assay. After removing free biotin-HPDP, S-nitrosylated proteins were purified on streptavidin-agarose gels. Eluates were immunoblotted with anti-actin antibody as described in "Methods."

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