Hrp conjugated anti rabbit igg h l

Proteins from 50 mg of liver samples were extracted, separated in gradient SDS-PAGE gels and electroblotted onto nitrocellulose membranes and western blot analysis was performed as described^{49 (link)}. Specific blotted proteins were detected with the corresponding primary rabbit antibody: anti-AMPK, anti-phospho-AMPK, anti-Akt, -anti-phospho-Akt (Cell Signaling Technology Inc. MA.; cat. number: #2532, #2535, #9272 and #9271, respectively); mouse anti-βactin (Sigma-Aldrich; cat. number: # A5316) and anti-Adaptin γ (Abcam, Cambridge, UK.;, cat. number #ab167153) followed by 1 h incubation with the corresponding secondary antibody: HRP-conjugated anti-rabbit IgG (H+L) or HRP-conjugated anti-mouse IgG (H+L) antibodies (Promega; cat. number W4011 and W4021, respectively). Specific protein bands were revealed using the enhanced chemiluminiscence detection system (Santa Cruz, Biotechnology Inc. CA, USA), in accordance with the manufacturer’s instructions. Images were visualized in an Autochemi-UVP Bioimaging System. Finally, bands were quantified by densitometry performed by ImageJ software (http://imagej.nih.gov/ij). Levels of specific proteins were normalized to actin levels for liver samples or adaptin levels for skeletal muscle samples.

Recombinant NS1 proteins from DENV-1-Nauru/Western Pacific/1974 (Accession: M23027.1), DENV-2-Thailand/16681/84 (U87411.1), DENV-3-Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (AY099336.1), DENV-4-Dominica/814669/1981 (AF326573.1), ZIKV-Suriname (AZS35340.1), and ZIKV-Uganda MR766 (AWF93629.1) produced in HEK293 cells were purchased from The Native Antigen Company (United Kingdom). Nunc Maxisorp plates (ThermoFisher Scientific) were coated with recombinant NS1 (50 μL/well of 2 μg/mL) in coating buffer (0.1M NaHCO₃) overnight at 4°C. Wells were washed three times with PBST (PBS supplemented with 0.05% Tween) and blocked with PBS supplemented with 5% skim milk (Sigma-Aldrich) for 1 h at RT. After washing the wells once with PBST, purified antibodies (50 μL of 1μg/mL) was added into the wells for 2 h at RT. Wells were washed three times with PBST, and 50 μL/well of peroxidase-conjugated anti-human IgG (1/5000 dilution, Sigma Aldrich) or HRP-conjugated anti-rabbit IgG (H+L) (1/5000 dilution, Promega) was added for 2 h at RT. After the wells were washed 3X with PBST, 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate system for ELISA (50 μL/well, Sigma) was added for ~ 5minutes, and then 1M HCL was added. The OD of the wells were determined at 450nm and 650nm (reference OD).

The following chemical reagents were used at the indicated concentrations: ISRIB (Tocris), 200 nM; dithiothreitol (DTT; Sigma-Aldrich), 5 mM; and poly(I:C), 15 μg (Invivogen). Cells were irradiated using a Spectrolink UV crosslinker at energy setting 200 J/m². The following primary antibodies were used: rabbit polyclonal anti-RPS10/eS10 (1:2000; Abclonal, A6056), rabbit polyclonal anti-ISG15 (1:5000; Cell Signaling Technology, 2743), rabbit polyclonal anti-ZNF598 (1:7000; Sigma-Aldrich, HPA041760), rabbit monoclonal anti-RPS20/uS10 (1:1000; Abcam, ab133776), rabbit monoclonal anti-phospho-eIF2α (Ser51) (1:2000; Cell Signaling Technology, D9G8) and mouse monoclonal anti-α-tubulin (1:10,000; Cell Signaling Technology, DM1A). The following secondary antibodies were used (at 1:5000 dilution): HRP-conjugated anti-rabbit IgG (H+L) (Promega, W4011) and HRP-conjugated anti-mouse IgG (H+L) (Promega, W4021). Cells were transfected using Lipofectamine RNAiMax (Thermo Fisher Scientific) for poly(I:C) transfections and with Lipofectamine 2000 (Thermo Fisher Scientific) for plasmid transfections. Cells were treated with indicated units of interferon-β for 16 h.