Anti ha antibody

Western blotting and reporter assay were performed as previously described [12 (link)–14 (link), 45 (link)–47 (link), 52 (link)]. For the Western blotting of virus particles, 340 μl of the culture supernatant was ultracentrifuged at 100,000×g for 1 h at 4 °C using a TL-100 instrument (Beckman), and the pellet was lysed with 1 × SDS buffer. For the Western blotting of transfected cells, the cells were lysed with RIPA buffer (50 mM Tris–HCl buffer [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche). The following antibodies for Western blotting: anti-His polyclonal antibody (OGHis; Medical and Biological Laboratories), anti-HA antibody (3F10; Roche), anti-FIV p24 Capsid antibody (PAK3-2C1; Santa Cruz Biotechnology); anti-alpha-tubulin (TUBA) antibody (DM1A; Sigma), and anti-VSVg antibody (P5DA; Roche). For FIV reporter assays, HEK293T cells were used for the target of infection. Ten microliter of the culture supernatant of transfected cells was inoculated into HEK293T cells in a 96-well plate (Nunc), and the firefly luciferase activity was measured by using the BrillianStar-LT assay system (Toyo-b-net) and the 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ procedures.

50 mL yeast cultures in S.D. media were harvested and disrupted using acid-washed glass beads [23 (link)]. The cell lysates were separated by SDS-PAGE and proteins were transferred to polyvinylidene fluoride membranes (Amersham Hybond-P 0.45, GE Healthcare Life Sciences, München, Germany). Chemiluminescence detection (ECL Chemostar, Intas Science Imaging Instruments, Göttingen, Germany) was carried out using a monoclonal mouse Anti-HA antibody (1:5000, Roche; cat. no. 11583816001, Mannheim, Germany), a horseradish peroxidase secondary goat anti-mouse antibody (1:5000, Jackson ImmunoResearch/Dianova; cat. no. 115-035-174, Hamburg, Germany), and the Clarity Western ECL substrate (Bio-Rad, Feldkirchen, Germany).

Preparation of chemically fixed cells for immunofluorescence and analysis of subcellular distribution of reporter proteins by wide-field and confocal microscopy were done as described previously [42 (link),54 (link)]. Nuclear labelling was performed with 4',6-diamidino-2-phenylindole (DAPI). The HA epitope tag was detected with a monoclonal anti-HA antibody coupled to FITC (dilution 1:50; Roche) whereas GlIscU was detected with a self-made antibody (dilution 1:300) followed by an anti-mouse antibody coupled to Alexafluor 594 (dilution 1:300; Molecular Probes). To avoid any possibility for cross reaction, co-labelling experiments for IFA were performed by incubating first with the anti-GlIscU antibody, followed by the AF594-conjugated anti-mouse secondary antibody, and direct detection of the HA epitope tag with a FITC-conjugated rat anti-HA monoclonal antibody as a final step.