Licor scanner

In order to perform in vitro translation, both the Wheat Germ Extract kit (Promega, Madison, WI, USA) and the FluoroTect™ GreenLys Labeling System (Promega, Madison, WI, USA) were used according to manufacturer’s instructions with the following modifications. Briefly, the reaction was performed in 25 µL containing 5 µg viroid RNA (specifically, (+) dimeric, (−) dimeric, (+) monomeric and (−) monomeric) and 2 µL of FluoroTect™. The reactions were carried out at 25 °C for 60 min, followed by an incubation at 30 °C for 60 min. The reactions were then terminated by the addition of RNase A (Promega, Madison, WI, USA). For PSTVd-derived translational analysis, 5 µL of the in vitro translation reactions were separated on a 12% SDS-PAGE gel and were then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, CA, USA). Anti- BODIPY™ FL rabbit IgG (ThermoFischer Scientific Inc, Waltham, MA, USA) at a dilution of 1:500 dilution was used to detect the translation according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), followed by a subsequent incubation with a 1:10,000 dilution of the IRDye 800CW donkey anti-rabbit-IgG polyclonal antibody (LI-COR). The proteins were subsequently visualized using an LiCOR scanner (LI-COR, Lincoln, NE, USA) at 700 nm.

Muscle tissues were homogenized in RIPA buffer containing a protease inhibitor cocktail (Santa Cruz, Dallas, TX) and phosphatase inhibitor (Research Product International, Mount Prospect, IL). Protein concentration of samples were determined by a standard BCA assay and the samples were subjected to standard western blot protocol, as described previously (Hartnett et al. 2015). Proteins that were transferred onto the membranes were immunoblotted using the following primary antibodies: AKT, phosphorylated AKT (Ser473), FoxO3, phosphorylated FoxO3 (Ser253), LC3, IRS1, AMPKα and phosphorylated AMPKα (Thr172) (Cell Signaling Technologies, Danvers, MA); GAPDH, Ubiquitin, pERK and ERK (Santa Cruz Inc., Santa Cruz, CA); MurF1 and Atrogin1 (GeneTex, Irvine, CA); and PGC1α (Millipore‐Sigma, St. Louis, MO). The secondary antibodies conjugated with Alex‐700 or ‐800 fluorescence were purchased from Invitrogen (Thermo Fisher Scientific Inc., Carlsbad, CA). The fluorescent signals of the blots were detected by LI‐COR scanner (LI‐COR Biosciences, Lincoln, NE) and quantified using LI‐COR Image Studio.

To determine the expression of proteins of interest, 30 μg of proteins in 5% SDS were separated on a polyacrylamide gel (4% stacking, 10% resolving gel) at 150 V for 70 minutes. The proteins from the gel were transferred to a polyvinylidene fluoride membrane at 0.35 Amp for 90 minutes. The transferred blots were blocked with 5–10 mL of Li-Cor blocking buffer (LI-COR Biosciences, Lincon, NE) for 1 hour and incubated overnight with primary antibodies (GAPDH Rabbit Mab, 1:2000 dilution, Cell Signaling Technology, Danvers, MA; CYP1A1 rabbit Mab, 1:200 dilution, Abcam, Cambridge, MA; CYP3A4 Mouse Mab. 1:200 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX; SOD1 Mouse Mab, 1:1500 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX; Catalase Mouse Mab, 1:1200 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX) at 4°C. After subsequent washing, the blots were incubated with corresponding secondary antibodies (1:10000 dilution, Goat anti-Mouse Mab, LI-COR Biosciences, Lincon, NE; 1:10000 dilution, Goat anti-Rabbit Mab, LI-COR Biosciences, Lincon, NE) for 1 hour at room temperature. The blots were scanned with Li-Cor Scanner (LI-COR Biosciences, Lincon, NE) and the densitometry data obtained from Image Studio Lite version 4.0 were used to calculate the fold expression of the proteins. GAPDH was used as an internal loading control to normalize the expression of sample proteins.