Irdye 800cw goat anti human igg

SDS-PAGE was performed under reducing conditions as previously described using a total of 4 μg antigen for subsequent Western blot analysis. After gel electrophoresis, the resolved N332 scaffolds (reducing conditions) were transferred to a polyvinylidene difluoride (PVDF) membrane by the use of a Trans-Blot Turbo transfer system (Bio-Rad). The membrane was blocked with 5% nonfat milk. The immobilized N332 scaffolds were detected with 6×His epitope mouse antibody (Thermo Fisher) at 1 μg/ml and PGT128 human IgG at 4 μg/ml, with IRDye 680RD anti-mouse IgG and IRDye 800CW goat anti-human IgG (LI-COR Biosciences) (diluted 1:10,000) used as secondary antibodies, respectively. The immunoblots were analyzed with an Odyssey Infrared Imaging System and Image Studio software (Li-COR Biosciences).

Blood obtained via tail bleeds were centrifuged to separate serum and frozen at −80 °C. C6/36 cells were pre-seeded at a concentration of 10⁵ cells per well in a 96-well plate. Sera were titrated out in RPMI with 2% FBS and PSG in tenfold serial dilutions and transferred onto pre-seeded cells (25 μL per well). Cells were incubated to infect for 2 h at 28 °C, 5% CO₂, before removing the inoculum and adding 200 μL of overlay media (2X M199 medium supplemented with 5% FBS, PSG and 2% carboxymethyl cellulose. Media was removed from the cells 48 h after infection and cells were fixed in 100 μL 80% ice-cold acetone in PBS at −20 °C for 30 min, then air-dried. For probing, plates were blocked with 100 μL KPL milk diluent/blocking solution in PBS-T at 37 °C for 1 h. Fifty microliters of purified hE16 [25 (link),26 (link)] antibody (1μg/mL) was added and incubated for 1 h at 37 °C to label for WNV E protein. Plates were washed 3 times in PBS-T before adding 50 μL of secondary antibody (IRDye 800CW goat anti-human IgG, LI-COR at 12.5 ng/well) and incubated at 37 °C for 1 h. After five PBS-T washes, plates were air-dried and scanned on Odyssey Clx reader with the following specifications: channel = 800, intensity = auto, resolution = 42 μm and focal length = 3mm. Resulting viral titers are shown as foci forming units per mL (FFU/mL) [27 (link)].

Cells were lysed in 3T3 lysis buffer (50 mm NaCl, 50 mm NaF, 30 mm sodium pyrophosphate, 25 mm HEPES, 2.5 mm EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X‐100; pH 7.5) supplemented with cOmplete™ EDTA‐free protease inhibitor cocktail (Sigma‐Aldrich). Protein concentrations were determined via Bradford assay. Equal concentrations of protein samples were separated via SDS–polyacrylamide gel electrophoresis (SDS/PAGE) and transferred onto nitrocellulose membranes for immunoblotting. Membranes were blocked in 5% (w/v) milk powder or 5% bovine serum albumin (w/v) in TBS‐T for 1 h prior to primary and secondary antibody incubations in the same solution. Primary antibodies used: anti‐PDE4D7 1 : 500 (Baillie Lab), anti‐VSV 1 : 5000 (Abcam, #ab1874), anti‐GAPDH 1 : 5000 (Abcam, #ab8245), anti‐DHX9 1 : 2000 (Abcam, #ab26271), anti‐FLAG 1 : 2000 (Abcam, #ab1257), anti‐phospho‐DHX9 1 : 200 (Baillie Lab), anti‐phospho‐PKA substrates 1 : 1000 (Cell Signalling, #9624S), anti‐GM130 1 : 1000 (Abcam, #ab52649), anti‐HSP90 1 : 1000 (Santa Cruz Biotechnology, #sc‐7947). Secondary antibodies used: IRDye® 800CW Donkey‐anti‐Rabbit IgG 1 : 5000 (LI‐COR, #926‐32213), IRDye® 680RD Donkey‐anti‐Goat IgG 1 : 5000 (LI‐COR, #926‐68074), IRDye 800CW Goat‐anti‐Human IgG 1 : 5000 (LI‐COR, #926‐32232), IRDye® 680RD Donkey‐anti‐Mouse IgG 1 : 5000 (LI‐COR, #925‐68072).