Goat anti mouse igg hrp antibody

Calf-thymus DNA was reconstituted in water 50 µg/ml and used to coat 96-well MaxiSorp plates overnight at 4 °C. The plates were washed with PBS, blocked with 1% casein buffer for 2 h at room temperature and subsequently washed with PBS. Mouse sera (1:30) or mouse anti-dsDNA antibody (1:100) were diluted in blocking buffer, transferred to the plates and incubated for 2 h at room temperature. The plates were washed with PBS with 0.2% Tween-20 and incubated with 1:5000 dilution goat anti-mouse-IgG-HRP antibody (BioRad) for 1 h at room temperature. Following washing with PBS with 0.2% Tween-20, TMB was added, the plates were incubated for 3 min and the reaction was stopped using sulfuric acid. DNA-specific IgG titers were determined by absorbance measurements at 450 nm. Endpoint dilutions were calculated using an absorbance cut-off of 0.05 as determined from the limit of detection determined for PBS-incubated wells (Graphpad Prism v.9.1, GraphPad Software Inc).

Proteins were loaded in equal amounts and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore). Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich). The following secondary antibodies were used as needed: a goat-anti-mouse-IgG-HRP antibody (Bio-Rad, Feldkirchen, Germany), a goat-anti-rabbit-IgG-HRP (Bio-Rad), or a goat-anti-sheep-IgG-HRP antibody (R&D Systems). Visualization was performed using the ChemoCam (INTAS, Homburg, Germany) after incubation with Clarity Western ECL Substrate (Bio-Rad).

Transfection with plasmids that expressed RABV proteins was performed using Metafectane reagent, following the manufacturers’ instructions (Biontex). We added 500 nM 17-AAG to the transfected cells 6 h after infection, and the incubation was terminated 48 h after transfection. The protein extracts prepared with the RIPA buffer were resolved on 10% PAGE-SDS gels to detect myc-tagged G, M, N, and P proteins. 3xFLAG-L protein was resolved on 8% PAGE-SDS gel. The insoluble protein was separated by centrifugation for 20 min at 14,000× g and the pellet was solubilized in 8M urea before electrophoresis. The proteins were detected using monoclonal antibodies specific for Flag (Sigma, F3165) and myc (Merck, MABE282). A goat anti-mouse IgG-HRP antibody was obtained from Bio-Rad (cat. no. 170-6516). The original pictures were taken with a CCD camera with the exposure time adjusted to avoid pixel saturation. The registered signals were within the linear response range of the camera.