Hrp conjugated goat anti rabbit igg

Mouse anti-puromycin antibody was obtained from Millipore. Rabbit anti-phospho-eIF2α (S51) and mouse anti-Myc antibody were obtained from Cell Signaling Technology. Mouse anti-HA and mouse anti-Flag antibody were purchased from MBL. Mouse anti-GFP antibody was obtained from ABclonal. Rabbit anti-beta (β)-actin antibody was obtained from Proteintech. HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG were purchased from Beyotime. Rabbit anti-VP3 antibody was prepared in our laboratory (Shen et al., 2016 (link)).
C16 (PKR Inhibitor) and GSK2606414 (PERK inhibitor) were purchased from APExBIO, and GCN2-IN-1 (GCN2 inhibitor) was purchased from MCE. These inhibitors were dissolved in dimethyl sulfoxide (Sigma) and configured to 10 mmol/L, which were diluted to working concentration with MEM when used. Sodium arsenite was purchased from Sigma. Poly(I:C) was purchased from Invivogen.

The DPV-CHv strain (GenBank: JQ647509) of the duck plague virus was isolated and preserved by the Research Center for Poultry Disease Control and Prevention, Sichuan Agricultural University. The 10-day-old duck embryos were purchased from a duck farm in Pixian County, Sichuan Province, and used to make DEF cells. Taking 12-well plates as an example, each test used 5.86 × 10⁵ cells. Mouse ANTI-FLAG monoclonal antibody and mouse anti-HA monoclonal antibody were purchased from MBL. Rabbit anti-pUL48 polyclonal antibody serum and rabbit anti-pUL47 polyclonal antibody serum were prepared and preserved by the research Center of Poultry Disease Prevention and Control, Sichuan Agricultural University. Rabbit anti-beta (β)-actin antibody was obtained from Proteintech. HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG were purchased from Beyotime.

Swine Testis (ST) and HIEC-6 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Billings, MT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and a 1% antibiotic antimycotic solution (Solarbio, Beijing, China) at 37 °C in a humidified atmosphere of 5% CO₂ [21 (link)]. ST and HIEC-6 cells were purchased from ATCC. The anti-PDCoV N rabbit monoclonal antibody was prepared by our laboratory. HRP-conjugated goat anti-rabbit IgG (A0208, Beyotime, Shanghai, China) was procured from Beyotime. GoTaq^® QpcR Master Mix (A600A, Promega, Madison, WI, USA) was acquired from Promega.

BmN-SWU1 cells were transfected with the bacmids vBm^WT, vBm^ie1-null, and vBm^{ie1-null-IE1(HA)}. At 24, 48, and 72 h p.t., the cells were lysed with western and IP cell lysis buffer (Beyotime), and the total proteins were harvested. After SDS-PAGE, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, USA), blocked with TBST containing 10% skim milk powder, and then incubated with rabbit anti-VP39 or anti-tubulin antibody for 1 h (Beyotime). After washing six times with TBST, the blots were incubated with HRP-conjugated goat anti-rabbit IgG (Beyotime). After washing six times with TBST again, the Western blot results were analyzed with the an ECL Western Blotting Detection System (Bio-Rad, Hercules, CA, USA).

For MAPK and NF-κB analysis, RAW264.7 cells were seeded in 6-well microplates at a density of 8 × 10⁵ cells per well for 16–18 h. To exclude the effects of residual LPS in rNFA49590 protein, the preparation was preincubated with 100 ug/mL polymyxin B (PmB, a specific inhibitor for LPS, INALCO, USA) at 37 °C for 2 h. Then 2, 4, or 8 μg/mL of rNFA49590 protein (with or without 100μg/mL PmB) or 100 ng/mL LPS (with or without 100μg/mL PmB) was added to the cell plate. At the 30, 60, and 120 min time points, whole-cell extracts were harvested using RIPA lysis buffer (strong) (CWBIO, Beijing, China) containing 1% protease and 1% phosphatase inhibitor cocktail. After the protein concentration was measured using a BCA protein assay kit, equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes as described before. Primary antibodies against p-ERK1/2 (1:1000, CST, Danvers, MA, USA), p-JNK (1:1000, CST, Danvers, MA, USA), p-P38 (1:1000, CST, Danvers, MA, USA), p-P65 (1:1000, CST, Danvers, MA, USA), and β-actin (1:4000, CST, Danvers, MA, USA) were used. Secondary antibodies included HRP-conjugated goat anti-rabbit IgG (1:1000, Beyotime, Shanghai, China) or HRP-conjugated goat anti-mouse IgG (1:4000, ZSGB-BIO, Beijing, China).