Goat pab to rabbit igg

Chemicals used include SB 204990 (Thermo Fisher Scientific, Paisley, UK), TOFA, C75, clofibrate, palmitic acid, and SC-236 (Sigma-Aldrich, Dorset, UK), PGD₂, PGI₂, and PGE₂ (Cambridge Bioscience, Cambridge, UK), and SC-51322, TG-4-155, and ER-819762 (Bio-Techne Ltd., Abingdon, UK), and all were reconstituted in dimethyl sulfoxide. TXA₂ (Cambridge Bioscience, Cambridge, UK) was reconstitute in ethanol. PGF_2α (Cambridge Bioscience, Cambridge, UK) and etomoxir and malonyl-CoA (Sigma-Aldrich, Dorset, UK) were reconstituted in E199 medium. Anti-β-actin mouse monoclonal antibody (MAb), anti-FASN goat polyclonal antibody (pAb), anti-COX2 goat pAb, and anti-ACC rabbit MAb (Abcam, Cambridge, UK) were used for Western blot analysis. Donkey pAb to goat IgG, goat pAb to mouse IgG, and goat pAb to rabbit IgG were also purchased from Abcam, Cambridge, UK.

Primary antibodies used were mouse monoclonal antibody (mAb) to chicken α-tubulin, rabbit mAb (IgG) to HMG-CoA (Cambridge Bioscience, Cambridge, UK), and rabbit polyclonal antibody (pAb) to squalene epoxidase (Sigma-Aldrich/Merck, Dorset, UK). The LEP100 hybridoma line (anti-LAMP-1; mouse IgG1 isotype) was obtained through the Developmental Studies Hybridoma Bank (DSHB) (University of Iowa, Iowa City, IA, USA). The HB3 hybridoma line (anti-gB; mouse IgG2b isotype) is an in-house-available hybridoma. Antibodies were purified in-house by a high-performance liquid chromatography (HPLC) method. Secondary antibodies used were goat pAb to rabbit IgG (Abcam, Cambridge, UK), goat anti-mouse IgG1-568 nm, and goat anti-mouse IgG2b-568 nm (Thermo Fisher Scientific, Paisley, UK).

IL-17A and IFN-α2 in the cell culture supernatants of CHIKV-infected NIH3T3 cells was measured by using a commercial ELISA kit (Abcam) following the manufacturer’s instruction. NIH3T3 cells were infected with CHIKV at 1 MOI in the presence of recombinant IL-17A. After 24 h, the cells were lysed in Laemmli sample buffer (Bio-Rad), and the proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked in 5% milk in Tris-buffered saline with Tween 20 (TBS-T) for 1h at RT and probed with mouse specific Rabbit primary antibody (Phospho-IRF-7, Cell Signaling; Phospho-IRF-5, Invitrogen; GAPDH, Abcam) in the ratio 1:1,000 at 4°C overnight in a rocker. After washing with TBS-T, horseradish peroxidase conjugated secondary antibody (Goat pAb to Rabbit IgG, Abcam; 1:5,000) was added for 1 h at RT. The membranes were washed and developed using SuperSignal West Pico Chemiluminiscence Substrate (Thermo Scientific) and images were acquired using a ChemiDoc MP system (Bio-Rad). Quantification of blot was performed by ImageLab.