Peroxidase conjugated mouse anti rabbit igg

C-proteasome analysis was performed in plasma by a self-developed ELISA assay, as previously described (16 (link)). Briefly, ELISA plates were coated with a mouse monoclonal antibody toward 20S proteasome-subunit α6 (Enzo Life Sciences, Farmingdale, NY, USA), and 20S purified proteasome in a concentration range of 0–100 ng/ml was used as calibration standard. An antiproteasome rabbit pAb (obtained from an expert research team) and then a peroxidase-conjugated mouse anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were applied for antigen detection. OD-values were determined at 450 nm. Every sample was tested in triplicate, and the mean of the values was reported.

Poly(I:C)-treated or DENV-2-infected CCL-125 cells were collected and lysed using lysis buffer (1 mM of DTT and 1× protease inhibitor in HEPES-NaCl-Glycerol-Triton X 100 (HNTG) buffer). The cell lysates were run on a 10% polyacrylamide gel. The proteins were transferred to a membrane (Millipore Immobilon-P, Merck Millipore, Burlington, MA, USA), which was incubated with anti-eIF2α (phospho S51) primary antibody (Abcam, Cambridge, UK) for 12 h. The peroxidase-conjugated mouse anti-rabbit IgG (Jackson Laboratory, Bar Harbor, ME, USA) secondary antibody was reacted with horseradish peroxidase (HRP) substrate (Merck Millipore, Burlington, MA, USA) Actin antibody (Jackson Laboratory, Bar Harbor, ME, USA) was used as a loading control.