IRDye 800CW

Proteins were separated by SDS-PAGE under reducing or non-reducing conditions. For immunoblotting, the proteins separated on SDS-PAGE were transferred onto nitrocellulose (Whatman) by wet blotting. The protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The recombinant NP and its N—and C-terminal fragments (rNP, rNP-N, and rNP-C) of University of Helsinki virus-1 (UHV-1) described in [29 (link)] were used as the antigen (1:1:1 pool, 1.5 μg of total protein per lane) when measuring anti-reptarenavirus antibodies by immunoblotting. The immunoblotting with snake sera in brief: The nitrocellulose membranes were blocked (3% skimmed milk and 1% bovine serum albumin [BSA] in TBS with 0.05% Tween-20 [T-TBS]) on an orbital shaker for 30 min at RT, incubated overnight at 4°C with snake sera (diluted (1:300) in T-TBS with 3% BSA), washed three times with T-TBS, incubated for 1 h at RT with either anti-IgM or -IgY antibody (both 1:2,000 dilution in blocking buffer), washed three times with T-TBS, incubated for 1 h at RT with IRDye 800CW-labelled donkey anti-rabbit (dilution 1:10,000 in blocking buffer, LI-COR Biosciences), and washed three times with T-TBS and once with PBS prior to recording the results, using an Odyssey Infrared Imaging System (LI-COR Biosciences). The immunoblottings with IgY and IgM as the antigens were done following a similar procedure, except that enhanced chemiluminescence with in-house reagents and recording on X-ray film (FUJI Medical RX) was used for recording the results with HRP-labeled anti-IgM and –IgY.

Whole cell lysates were prepared at different time points after infection and transfection using RIPA lysis buffer (Sigma-Aldrich, R0278) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime, ST506-2) in accordance with the manufacturer’s instructions. The total protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, 23225). Equal amounts of total proteins were analyzed with SDS-PAGE and then transferred onto nitrocellulose membranes (Pall, 66485). After blocking with 5% skim dry milk for 1 h at 37°C, the membranes were incubated with different primary antibodies for 6–8 h at 4°C, and followed by incubation with IRDye 800CW goat anti-mouse lgG (H+ L) (1:10,000) (926–32210; LiCor BioSciences) and IRDye 680RD goat anti-rabbit lgG (H+ L) (1:10,000) (926–68071; LiCor BioSciences) at 37°C for 1 h, and thereafter the blots were visualized using an Odyssey infrared imaging system (LiCor BioSciences). Quantification of band intensities by densitometry was carried out using the Image J software.

After the indicated treatments, cells were washed with ice-cold PBS and lysed in RIPA buffer (#sc-24948, Santa Cruz Biotechnology) or cell lysis buffer (#9803, Cell Signaling) containing protease and phosphatase inhibitors. Equal amounts of protein quantified by Bio-Rad Protein Assay Dye (#5000006) were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with the designated antibodies (1:1000 dilution). GAPDH (1:5000 dilution) was used as a loading control. The membranes were scanned using a LI-COR Odyssey IR imaging system capable of detecting antigen-antibody complexes labelled using fluorescent goat anti-rabbit (IRDye 680 RD; 1:10,000 dilution) or goat anti-mouse (IRDye 800CW; 1:10,000). Phosphoproteins were probed first, followed by stripping and re-probing the same blot with the total antibody and loading control. Quantification of bands was done using Image Studio Lite software provided with the LI-COR imaging instrument or Image J. Phosphoprotein band intensities detected by Western blotting were normalized to the levels of total protein detected by respective antibodies. All others were normalized to GAPDH. Experiments were repeated at least 3 times and a representative Western blot from one experiment is shown in the figures.