Anti gapdh ga1r

Western blot analyses were performed essentially as previously described (44 (link)). To detect the relative level of MDV infection, mouse mAb H19 (45 (link)) was used at 1:10,000 dilution to detect MDV pp38. To detect RLORF4 tagged with mRFP, anti-mRFP polyclonal antibody (ab62341; Abcam, Cambridge, MA, USA) was used at 1:2000 dilution. Anti-Flag M2 mAb (F1804, Sigma-Aldrich) were used at the manufacturer's recommended dilutions to detect 3 × Flag-tagged pICP27 (UL54). For protein loading controls, anti-GAPDH (GA1R; Thermo Scientific) and anti-β-Actin (ACTNO5; Abcam) mAb were used at their recommended dilutions. Secondary anti-mouse or rabbit IgG-peroxidase conjugate was purchased from GE Healthcare (Piscataway, NJ, USA). The SuperSignal West Pico Chemiluminescent Substrate kit from Thermo Fischer Scientific (Rockford, IL, USA) was used to detect antigens utilizing the manufacturer's instructions. Images were obtained using a FluorChem R imaging system (ProteinSimple, CA, USA) in 8-bit format. Protein bands were quantified using ImageJ software (version 1.6) for densitometric analysis by comparing the relative ratios of viral protein to GAPDH using the technique described on the ImageJ website (https://imagej.nih.gov/ij/docs/).

Protein extracts were prepared from cells by using RIPA buffer, and the protein concentration was measured by protein assay kit (Bio-Rad, USA). Protein extracts were denatured in sample buffer and subjected to SDS-PAGE gel electrophoresis. The electrophoretic proteins were then transferred to the nitrocellulose (NC) membrane. Nitrocellulose membranes were blocked in 5% skimmed milk and probed with primary antibodies. NC membrane were then washed and incubated with HRP-conjugated secondary anti-rabbit IgG or anti-mouse IgG at room temperature in TBST containing 5% milk for 1 h. After extensive washes in TBST, the signals were visualized by the enhanced chemiluminescence system as described by the manufacturer (Millipore, Germany) in conjunction with in LAS-4000 image analyzer (GE Healthcare, Japan). The immunoblotting signals from anti-Beta-actin (BA3R, Thermo Fisher Scientific, USA) or anti-GAPDH (GA1R, Thermo Fisher Scientific, USA) antibodies were used as a loading control.

Liver tissues and cells lysates were prepared with RIPA buffer containing 1× EDTA/proteinase-phosphatase inhibitor cocktail (Pierce). The lysate supernatant was stored at −80 °C until used for immunoblotting. Protein extracts were separated by SDS-PAGE electrophoresis and blotted onto nitrocellulose membrane. Blots were incubated overnight with the following primary detection antibodies: anti-α-SMA EPR5368, (1:2000, Abcam); anti-GAPDH GA1R and anti-β-actin (1:10000 ThermoFisher Scientific). The blots were then incubated with IRDye^® IgG secondary antibody conjugates (Li-Cor) and then revealed using an Odyssey^® gel documentation system following the manufacturer’s instruction (Li-Cor).