Horseradish peroxidase conjugated anti mouse or anti rabbit igg antibodies

SeV-infected cells were lysed with modified radioimmunoprecipitation buffer containing a protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland). Equal amounts of protein (10 µg) were subjected to electrophoresis using 10% sodium dodecyl sulfate–polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, washed with Tris-buffered saline containing 0.05% Triton X-100, and incubated with BlockingOne solution (Nacalai Tesque, Kyoto, Japan) for 60 minutes. Anti-HA tag (1:500; Cell Signaling Technology) or anti-SeV (1:2,000; MBL) was used as the primary antibody. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega, Madison, WI) were used as the secondary antibody. Signal Booster (Beacle, Kyoto, Japan) was used for diluting Anti-HA tag antibody and the secondary antibody. As a control, β-actin was detected with an anti-β-Actin pAb-HRP-DirecT (1:2,000; MBL). Blots were visualized using Chemi-Lumi One L (Nacalai Tesque).

For protein isolation from total cellular fractions, frozen skeletal muscle were homogenized in liquid nitrogen and incubated in the lysis buffer (Cell Lysis Buffer, Cell Signaling, Beverly, Massachusetts) supplemented with 0.1 mg PMSF and a 1:100 dilution of protease and phosphatase inhibitor cocktails (Sigma, St. Louis, Missouri). Homogenization was repeated, samples were centrifuged for 10 min at 14 000 g, and the supernatants were used for Western blot as described previously [10 (link)–12 (link)] with the antibodies against PTEN-induced putative kinase 1 (PINK1) (Abcam, Cambridge, Massachusetts) and actin (Sigma, St. Louis, MO). The complexes that formed were detected with horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG antibodies (Promega, Madison, Wisconsin) using chemiluminescent reagents (SuperSignal, Pierce, Rockford, Illinois). An oxidative protein carbonylation assay was performed as described previously [12 (link)]. Where indicated, the resultant band images were scanned and analyzed (Fujifilm Image Gauge Version 2.2 software, Fuji, Tokyo, Japan). For protein carbonylation, summarized band intensities for 2 major bands (~50 kDa and ~40 kDa) were normalized by the mean value of 1 HC sample set to 1 and presented as AU. For PINK1 protein content, data were normalized to the density of actin bands and presented as AU.