Protease phosphatase inhibitors cocktail

TriEx Sf9 cells co-infected with recombinant baculoviruses containing PRRSV M, N, E, and GP5 gene were harvested at 72 h after infection. Cells were lysed with a lysis buffer containing 0.01 M Tris-HCl, 0.14 M NaCl, 0.025% NaN3, 1% Triton X-100, and protease/phosphatase inhibitors cocktail (Thermo Scientific, Rockford, IL). Cell lysates and purified VLPs were subjected to SDS-PAGE gel electrophoresis (Novex by Life Technologies, Carlsband, CA) and transferred to a nitrocellulose membrane (Life Technologies, Gaithersburg, MD). The membrane was then blocked for 1 h by rocking slowly at room temperature with 5% (w/v) milk powder in PBS plus 0.05% Tween 20 (PBST). Next, primary antibody, monoclonal mouse anti-HA antibody (Sigma-Aldrich, St. Louis, MO) diluted 1:5,000 in blocking buffer 5% (w/v) milk powder in PBST was added to the membrane and incubated overnight at 4 °C on the rocker. The secondary antibody, goat anti-mouse IRDye (LI-COR, Lincoln, NE) diluted 1:10,000 in PBST was added to the membrane and incubated for 1 h rotating at room temperature. Bands were visualized using the ODESSY Infrared Imaging System (LI-COR, Lincoln, NE).

Frozen heart tissue (without the atria) was ground up with a mortar and pestle under liquid nitrogen. For total protein extraction, heart tissue was homogenized in T-PER^® tissue protein extraction reagent containing protease/phosphatase inhibitors cocktail (Thermo Scientific, Rockford, IL, United States) and 100 mM Phenylmethanesulfonyl fluoride (PMSF) following the manufacturer’s instruction. The extraction of nuclear/cytoplasmic protein was performed using NE-PER^® nuclear and cytoplasmic extraction reagents (Thermo Scientific, Rockford, IL, United States) according to manufacturer’s instruction. Protein concentrations were measured by Bradford protein assay (Bio-Rad Laboratories, Philadelphia, PA, United States) using bovine serum albumin (BSA) as standards.

Primary HUVECs at passage 2 were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan) and maintained in HUVEC growth medium (EBM-2, Lonza) with full supplements (2% fetal bovine serum, 0.4% human fibroblast growth factor-2, 0.1% vascular endothelial growth factor, 0.1% R³-insulin-like growth factor-1, 0.1% human epidermal growth factor, 0.04% hydrocortisone, 0.1% ascorbic acid, 0.1% GA-1000). Only passages 3 to 6 cells were used. Cells were treated with chemical inhibitors for 30 min before addition of inflammatory cytokine. At the indicated time point, cells were lysed with ice-cold lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% CHAPS, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO₄) and protease/phosphatase inhibitors cocktail (Thermo Scientific) immediately.