Rabbit anti β actin antibody 13e5

The cells were lysed in a buffer containing 125 mm Tris-HCl, pH 6.8, 5% SDS, 25% glycerol, 0.1% bromphenol blue, and 10% β-mercaptoethanol and boiled for 5 min. The cell lysates were then separated on a 10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride microporous membrane (Millipore). FluA-NP 4F1 (SouthernBiotech), a goat anti-influenza A viral NS1 antibody (vC-20, Santa Cruz Biotechnology, Inc.), a rabbit anti-firefly luciferase polyclonal antibody (MBL, Nagoya, Japan), and a rabbit anti-Renilla luciferase polyclonal antibody (MBL) were used as primary antibodies to detect their respective proteins. A rabbit anti-β-actin antibody (13E5, Cell Signaling) was used as an internal control. The secondary antibodies, horseradish peroxidase-conjugated goat anti-mouse IgG (SouthernBiotech), donkey anti-goat IgG (sc-2020, Santa Cruz Biotechnology), or goat anti-rabbit IgG (KPL), were used as appropriate. The signals were detected using Western Lightning ECL Pro (PerkinElmer Life Sciences). Signal intensities were measured using ImageJ software, and the protein levels of firefly and Renilla luciferase were normalized to that of β-actin.

Western blot analyses were performed as described previously 29. Briefly, proteins in the cell lysate were separated on 10% polyacrylamide gels, followed by transfer onto a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany). The membrane was hybridized with a mouse monoclonal anti‐c‐Myc antibody (9E10; Wako) and a rabbit anti‐β‐actin antibody (13E5; Cell Signaling Technologies, Danvers, MA, USA). The membrane was visualized using ImmunoStar LD (Wako).