Hrp coupled secondary antibody

Cells were lysed for 15 min at 4 °C in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P40) supplemented with protease inhibitors (CompleteTM protease inhibitor mixture, Roche, Rotkreuz, Switzerland). Supernatants were collected, and the protein concentration was determined with a BCA assay (Sigma, Deisenhofen, Germany). Equal amounts of protein samples were separated on 8% Tris/glycine gels and then transferred to nitrocellulose membranes (GE Healthcare, Uppsala, Sweden). Subsequently, the membranes were incubated with primary antibodies and HRP-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania, USA). Chemiluminescence was measured, using an imager and the software Fusion (Vilber Loumat).

Samples were subjected to SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat dry milk in TBST (50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.05% Tween-20) and incubated with primary antibodies as indicated. Antibodies used were: mouse anti-c-Myc 9E10 (1:1000, Thermo Fisher Scientific, Waltham, MA), mouse anti-FLAG (1:1000, Sigma Aldrich, St. Louis, MI) and mouse anti-actin (1:2000, Merck Millipore, Burlington, MA). Primary antibodies were detected using HRP coupled secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, Cambridge House, UK). Chemiluminescent signal was developed using Clarity and ClarityMax Western Blotting Substrates (BioRad Laboratories Inc., Hercules, CA) followed by detection with a FujiFilm ImageQuant LAS-4000 detector (GE Healthcare, Chicago, IL).

Protein concentration was measured using the bicinchoninic acid (BCA) method (Thermo Scientific). Subsequently, 20 μg of cell lysate was denatured with sample buffer (62.5 mM Tris–HCl pH6.8, 2% (v/v) SDS, 50 mM DTT, 10% (v/v) glycerol), subjected to 4%–20% gradient SDS-PAGE, and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked for 1 h with 5% (w/v) dried skimmed milk in TBS-T buffer (25 mM Tris, 150 mM NaCl, and 0.1% (v/v) Tween-20, pH 7.4), and incubated with the following primary antibodies: mouse anti-GFAP (Sigma-Aldrich; 1:50,000), rabbit anti-Iba1 (Wako; 1:10,000), mouse anti-β-actin (Sigma-Aldrich; 1:20,000), anti-NSE (1:1000, Dako), or anti-GAPDH (1:40000, GeneTex), at 4°C overnight. Then, membranes were incubated with the corresponding HRP-coupled secondary antibodies (Jackson Immunoresearch; 1:10,000) for 1 h at room temperature, and the bands were visualized using the Super Signal West Pico chemiluminescent substrate (Thermo Scientific). The immunoreactive signals were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Iba1 and GFAP values were normalized to β-ACTIN values. NSE results, due to molecular weight, were normalized to GAPDH values.

In-gel activity of complex V was determined as previously described^{39 (link)}. In brief, a gel strip was cut from the BN-PAGE gel and incubated for 30 min in complex V calibration buffer (35 mM Tris, 220 mM glycine pH 8.3). After this, the gel was transferred to complex V staining buffer (35 mM Tris, 220 mM glycine pH 8.3, 14 mM MgSO₄, 0.2% (w/v) Pb(NO₃)₂, 8 mM ATP) and incubated at 30 °C until bands appeared. TCA protein precipitation was performed according to standard procedure. SDS-PAGE, urea-PAGE, and western blotting were performed according to standard protocols. Signals were detected using HRP-coupled secondary antibodies (used in 1:10,000 dilution, Jackson ImmunoResearch, code number 111-035-144) and enhanced chemiluminescence system (GE Healthcare). No other image processing, other than cropping, scaling, and contrast adjustment using Adobe Photoshop CS6 and Adobe Illustrator CS6 was applied.

Dissected LAL muscles were homogenized in RIPA buffer (150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) containing a cocktail of protease inhibitors (Halt™ Protease Inhibitor Cocktail 100X; #78430, Thermo ScientificTM) along with 2 mM Na₃VO₄. For immunoblotting, proteins were separated in 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Scientific), and probed with rabbit anti β-catenin (1:1000, Millipore), rabbit anti nAChR γ-subunit (1:1000, Invitrogen), or mouse anti GAPDH (1:1000; Santa Cruz Biotechnologies). Bound antibodies were visualized using the respective HRP-coupled secondary antibodies (Jackson ImmunoResearch Laboratories) developed using a chemiluminescence reagent kit (Perkin Elmer).