Mouse anti tubulin

Cells were lysed at 4 °C in CHAPS lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 2 mm EDTA, 1% (w/v) CHAPS (Sigma-Aldrich), and protease and phosphatase inhibitor mixtures (Roche Applied Science)). The lysates were cleared by centrifugation (15,000 × g for 15 min), resolved on a 12% SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The antibody dilutions used were rabbit anti-FLAG (1:1,000; Sigma, F7425), rabbit anti-HA (1:10,000; Sigma, H6908), mouse anti-tubulin (1:10,000; Millipore, 05-829), rabbit anti-YFP/GFP (1:25,000; Abcam, ab290), and anti-SERCA (1:1,000; Calbiochem, 564702). Purified proteins were resolved on Novex 12% Tris/glycine gels (Invitrogen) in the absence of reducing agent and stained with Imperial stain (Pierce) or Coomassie Blue (R-250).

To extract proteins, adult heads were dissected and frozen on dry ice, and then homogenized in Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris-Cl pH 6.8, 0.02% bromophenol blue, 10% beta-mercaptoethanol). Samples were heated at 95°C for 5 min and loaded onto a SDS-PAGE gel. Gels were run first at 80 volts for 20 min, then 100 volts for the remainder of the time and transferred onto nitrocellulose membranes (Bio-Rad) for one hour at 100 volts. Membranes were washed for 5 min in TBST (20 mM Tris (pH 7.6), 136 mM NaCl, 0.2% Tween-20), and blocked in 5% low-fat milk in TBST solution for one hour. Membranes were incubated overnight with primary antibody in TBST with 5% milk at 4°C, washed three times for 10 min in TBST and incubated in horseradish peroxidase-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch) at room temperature in TBST with 5% milk for 2 hr. Membranes were washed three times for 10 min in TBST and once for 10 min in TBS. Enhanced chemiluminescence (Thermo SuperSignal WestPico) was used to develop the blots. Primary antibodies used were guinea pig anti-Loaf (1:1000, Proteintech) and mouse anti ß-tubulin (1:10,000; Sigma, T4026).

Persistently VSBV-1 infected Vero cells were fixed with 80% acetone, followed by rehydration and blocking with milk power. Cells were incubated for 2 h at 37°C with a bornavirus-reactive serum sample from a human patient (1:500) and mouse-anti-ß-tubulin (1:1000, Sigma Aldrich, Missouri, USA). Following subsequent washing with PBS, cells were incubated (1 h, 37°C) with anti-human-Alexa-488 (1:1000 in PBS, Fisher Scientific, New Hampshire, USA) and anti-mouse-Alexa-568 (1:1000 in PBS, Fisher Scientific, New Hampshire, USA) as secondary antibodies as well as Hoechst33342 (1 µg/ml) to make the nuclear chromatin visible. The specimens were mounted on coverslips and z-stack images (z-step size 0.35 µm) were acquired by confocal laser scanning microscopy (Leica DMI 6000 TCS SP5). Images were processed by using the Fiji, ImageJ (v1.52 h), distribution package [25 (link),26 (link)].