Odyssey blocking solution

To assess whether the various cytokines secreted from the activated microglia are caused by NF-κB activation due to GBP2 expression, an ICW assay was conducted using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA) according to the manufacturer’s instructions. Briefly, BV2 cells were seeded at 1 × 10⁴ cells/mL for 24 h in 96-well plates (Falcon™, Cat# 161093, BD Biosciences, San Jose, CA, USA; Nunc™, Cat# 161093, Thermo Fisher Scientific, Waltham, MA, USA), fixed with 3.7% formaldehyde for 20 min at room temperature (RT), and then blocked with LI-COR Odyssey Blocking Solution (LI-COR Biosciences) for 90 min. The cells were incubated at RT overnight with NF-κB pre-mixed with a mouse IgG antibody against β-actin (1:500 dilution, Santa Cruz Biotechnology, Dallas, TX, USA). After five washes with 0.1% PBST, the cells were stained with a goat anti-mouse IgG IRDye™ 680 antibody (1:800 dilution, LI-COR Biosciences) at RT for 1 h. The microplates were scanned using the Odyssey CLx Infrared Imaging System (LI-COR Biosciences), and the integrated fluorescence intensities representing the protein expression levels were acquired using the software provided with the imager station (Odyssey Software Version 3.0, LI-COR Biosciences). The relative amount of protein was obtained by normalizing to endogenous β-actin in all experiments.

Protein was extracted from differentiated myoblasts using NCH buffer (4% sodium dodecyl sulphate, 4 M urea, 150 mM Tris) diluted 1:1 with RIPA (Radio-Immunoprecipitation Assay) buffer (Sigma) containing a complete protease inhibitor cocktail (1:100, Roche). Samples were collected and boiled for 3 minutes and 30 μl of each sample was run on a NuPAGE Novex 3–8% Tris-Acetate Gel, with constant voltage of 150 V for 1 hour, before transfer to a nitrocellulose membrane at 300 mA for 2 hours. The membrane was blocked in Odyssey blocking solution (LI-COR Biosciences) for 60 min, before incubation with rabbit anti-dystrophin (1:2000, Fisher Scientific) or rabbit anti-FLAG-tag (1:2000, Sigma) and mouse anti-beta-actin (1:4000, Invitrogen) overnight at 4 °C. The membrane was washed with PBS containing 1% Tween 20 (PBST) before incubation with IRDye 680 RD goat anti-rabbit and IRDye 800CW goat anti-mouse 2nd antibodies (1:15000, LI-COR Biosciences) for 1 hour at room temperature. The fluorescent image was acquired by Odyssey Clx infrared imaging system (LI-COR Biosciences) using image studio software 3.1.4.

Cell lysates were obtained in lysis buffer containing Halt Protease & Phosphatase Single-Use Inhibitor Cocktail (Thermo Scientific), 20 µg of protein were loaded on 12% polyacrylamide gel and then transferred on nitrocellulose membrane. The membrane was blocked with Odyssey blocking solution (LI-COR, Lincoln, Nebraska) at room temperature for 1 h and followed by an incubation overnight with primary antibodies p53 DO-1 mouse monoclonal IgG (1:5,000, Santa Cruz Biotechnology) and Cdk2 rabbit polyclonal IgG (1:10,000, Santa Cruz Biotechnology) at 4 °C. The membrane was then incubated with secondary antibodies Goat anti-mouse IgG (1:5,000, IRDye® 680RD, LI-COR) and Goat anti-rabbit (1:10,000, IRDye® 800CW, LI-COR) at room temperature for 1 h in dark. Odyssey detection system (CLx, LI-COR) was used to visualize the immunoreactive bands. Image J software was used to quantify the density of the bands.

Proteins separated by electrophoresis as above were transferred to nitrocellulose membranes which were blocked with Odyssey blocking solution (LI-COR). After blocking, membranes were incubated overnight at 4°C with primary antibodies: mouse anti-ICP4 MAb (1:1500); mouse anti-ICP0 MAb (1:1500); mouse anti-VP5 MAb (Virusys) (1:1500); mouse anti-ICP8 MAb (Abcam) (1:1500) and rabbit anti-ᵞ-tubulin PAb (Sigma) (1:2000); diluted in blocking solution. Membranes were washed three times with 0.05% Tween in PBS and incubated with goat anti-mouse IgG Dylight 680 (Pierce Biotechnology) or goat anti-rabbit IgG Dylight 800 (Pierce Biotechnology) in blocking solution for 1 hr at RT in the dark. Visualisation of protein bands was performed using the LI-COR Odyssey Infrared Imaging System.