Sg substrate kit

Proteins were extracted using RIPA buffer (Sigma), supplemented with protease (1:1000, Sigma) inhibitor cocktails, after 30 min incubation on ice. Protein concentrations were measured using BCA assay, with BSA for the standard curve, and 50 μg of protein were resolved on 12% NuPAGE gels (Invitrogen) and transferred using a semi-dry transfer system onto PVDF membranes (Millipore). Non-specific binding sites were blocked with 10% milk TBS-T solution for 1 h at room temperature, then probed overnight at 4 °C with the respective primary antibodies: anti-SAMHD1 (Abcam), anti-SOX11 (Sigma), or anti-Cyclin D1 1:1000 in 5% milk or 5% BSA in TBS-T. Membranes were then washed in TBS-T and probed with secondary antibodies (HRP-conjugated anti-rabbit or anti-mouse; GE Healthcare). Blots were developed using Supersignal West Pico (Pierce) and visualized using LiCor machine. For re-probing of membranes with anti-actin (Sigma) or anti-GAPDH (Cell Signaling) (1:5000 in 5% milk in TBS-T, Sigma), HRP was blocked using the SG substrate kit (Vector Labs). Analysis was done using Fiji-ImageJ software.

Two hours after electrical or sham stimulation, rats were re-anesthetized with a mixture of Zoletil (25 mg/kg) and xylazine (10 mg/kg) and perfused with 500 ml 4% paraformaldehyde in 0.1 M PB (pH 7.4). The brain stem containing the CN was harvested, post-fixed with the same fixative for 2 h, and stored in PB containing 30% sucrose. Tissue blocks were cut transversely into 30-μm-thick serial sections and divided in order into four sets. One of the four serial sections were treated with 1% H₂O₂ and blocked with 5% normal goat serum in 0.1 M PB containing 0.2% Triton X-100 for 2 h. They were incubated in rabbit anti-c-Fos (1:2000, Calbiochem, San Diego, CA, USA) antibody at 4°C for 48 h. After several washes, sections were processed with biotinylated goat anti-rabbit antiserum (Vector, Burlingame, CA, USA) for 2 h at room temperature, and processed with avidin-biotin-horseradish peroxidase (HRP) complex (ABC kit, Vector), and visualized with a Vector® SG Substrate Kit. Finally, they were mounted onto gelatinized slides and their images were captured with a digital camera (Nikon, D1X, Tokyo, Japan) through a light microscope to measure c-Fos-LI cells in the CN.