Protease inhibitor mix g

NL, using 10 mL of sterile isotonic saline (0.9% NaCl, Braun, Melsungen, Germany) per nostril, was performed as described previously [32 (link)]. Immediately after collection, NL fluid was either aliquoted with and without protease inhibitor (PI) (Protease Inhibitor Mix G, SERVA Electrophoresis GmbH, Heidelberg, Germany) or centrifuged for 7 min at 400 rpm. Supernatants were aliquoted with and without PI and frozen at −70°C. For cytological analysis, 5 mL of NL was added to 0.5 mL fetal calf serum (FCS, Biochrom AG, Berlin, Germany). The suspension was centrifuged for 7 min at 400 rpm. Supernatant was discarded leaving 1 mL for resuspension of the cell pellet. 100 μL of FCS was added.

Hetero-dimer formation of the Hsp90 isoforms was assessed in yeast strains in which one Hsp90 isoform had a C-terminal GFP-tag and the other isoform had a C-terminal 6HA-tag. Tagging of the endogenous HSP90 isoforms was performed according to the protocol provided by Janke et al.^{45 (link)}. Cells were lysed in 50 mM Tris, pH 8, 20 mM NaCl and 0.15% NP40 and protease inhibitor Mix G (Serva). Co-immunoprecipitations were performed by incubating cleared lysates with GFP-Trap_A agarose (Chromotek) or Anti-HA agarose (Sigma) for 30 min. Beads were washes four times with lysis buffer and proteins were eluted by boiling the agarose for 5 min in 4× SDS buffer (NUPAGE). The following primary antibodies were used to probe the membrane: antiyeast Hsp90 polyclonal antibody (Pineda Antibody Service^{48 (link)} at a concentration of 1:30,000) anti-PGK1 monoclonal antibody (Novex, cat. no. 459250 at a concentration of 1:15,000), anti-HA monoclonal antibody (Sigma, product number H9658, at a concentration of 1:5000) and anti-GFP antibody (Roche, cat. no. 11814460001, at a concentration of 1:1000). The secondary antibodies were anti-mouse and anti-rabbit IgG-peroxidase conjugated antibodies (Sigma-Aldrich, cat. nos. A9044 and A0545, respectively, both used at a concentration of 1:20,000). Image acquisition was performed with an ImageQuant LAS4000 (GE Healthcare).

Cells grown to 60% confluency were lysed using RIPA buffer supplemented with phosphatase and protease inhibitors (Protease Inhibitor Mix G and Phosphatase Inhibitor Mix I, Serva, Heidelberg, Germany). Samples were separated by SDS-PAGE on 12% polyacrylamide gel and then transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking in Tween-PBS with 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) for at least 1.5 h, membranes were incubated with GM3 synthase and GM2/GD2 synthase antibody (1:2000; Santa Cruz sc-365329 and sc-376505, Dallas, TX, USA), or β-actin (1:2000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After washing, membranes were incubated with anti-mouse m-IgGκ BP-HRP (Santa Cruz sc-516102, Dallas, TX, USA) for 1 h. Immunocomplexes on the membranes were visualized with ECL Western Blotting Detection Reagents (Cell Signaling Technology) using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Virus infected cells were incubated with lysis buffer (20 mM Tris, pH
7.5, 100 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 1% protease
inhibitor mix G [Serva, Heidelberg, Germany], 1 mM dithiothreitol [DTT]) for
15 min on ice. After centrifugation at 13,000 rpm at
4 °C, supernatants were complemented with SDS page
sample buffer55 (link) and incubated at 95 °C.
Proteins were separated in SDS-PAGE gels (15%), and transferred to
nitrocellulose membranes. Antibodies for detection of NEP and NS1 were a gift
from Thorsten Wolff and Christina Ehrhardt respectively. The commercial
antibodies for detection of GFP and tubulin were purchased from Santa Cruz
Biotechnology (GFP) and Sigma-Aldrich (tubulin).

Cells grown at 50% confluency were lysed in RIPA buffer which was supplemented with phosphatase and protease inhibitors (Protease-Inhibitor Mix G and Phosphatase-Inhibitor-Mix I, Serva, Heidelberg, Germany). The cell lysates were clarified by centrifugation, separated by SDS-PAGE, blotted to 0.45 μm nitrocellulose membranes, blocked in 5% nonfat milk in PBS, and probed with primary antibodies. Fluorescent labeled anti-rabbit IRDye 700 and anti-mouse IRDye 800 secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) were used for visualization using an Odyssey infrared imaging system (LI-COR Biosciences). The rabbit phospho-p44/42 MAPK (Erk1/2), rabbit phospho-Akt (Ser473), mouse p44/42 MAPK (Erk1/2), and rabbit AKT primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The mouse p120RasGAP antibody was obtained from ECM Biosciences LLC (Versailles, KY, USA).