Alexa fluor 594 conjugated donkey anti rabbit igg

The rabbit monoclonal antibodies (mAb) against phospho-eIF2α (Ser51) and eIF2α were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse mAb against beta-actin was obtained from Medical and Biological Laboratories (MBL, Nagova, Japan). Antibodies against TIA1 (goat polyclonal), G3BP1 (rabbit polyclonal), TIAR (goat polyclonal), and eIF3η (goat polyclonal) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Rabbit mAb against NF-κB p65 and phospho-NF-κB p65 were purchased from Cell Signaling Technology. Mouse mAb against PRRSV nucleocapsid (N) protein was prepared by our laboratory. Alexa Fluor 594-conjugated donkey anti-goat IgG, Alexa Fluor 594-conjugated donkey anti-rabbit IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG, Alexa Fluor 488-conjugated donkey anti-mouse IgG, and Alexa Fluor 647-conjugated donkey anti-mouse IgG were also obtained from Santa Cruz Biotechnology, Inc. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit, HRP-conjugated goat anti-mouse, and HRP-conjugated donkey anti-goat were purchased from Medical and Biological Laboratories (MBL).

IPI-2I cells (porcine intestinal epithelial cells), BHK-21 cells, and HeLa cells were obtained from the China Center for Type Culture Collection (Wuhan, China). LLC-PK1 cells were acquired from the American Type Culture Collection (ATCC CL-101; Manassas, VA, USA). IPI-2I, BHK-21, and LLC-PK1 cells were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (Invitrogen, USA). HeLa cells were grown in RPMI 1640 medium with 10% fetal bovine serum. These cells were maintained in 5% CO₂ at 37 °C. PDCoV strain CHN-HN-2014 (GenBank accession no. KT336560)^{10 (link)} and TGEV strain WH1 (GenBank accession no. HQ462571) were isolated in 2014 and 2010, respectively, in China. Mouse monoclonal antibodies against TGEV M, TGEV N or PDCoV S, PDCoV N were created in-house. APN antibody was purchased from ABclonal (China). An anti-Flag rabbit polyclonal antibody (MBL, Japan), Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Santa Cruz, USA), and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Santa Cruz, USA) were used for indirect IFA. Horseradish peroxidase-conjugated goat anti-mouse antibody (Beyotime, China) and horseradish peroxidase-conjugated goat anti-rabbit antibody (Beyotime, China) were applied in western blots.

Porcine kidney (PK-15) cells and HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum in a humidified incubator with 37°C/5% CO₂. TGEV strain WH-1 (GenBank accession no. HQ462571) was propagated and titered in PK-15 cells. Recombinant VSV-expressing green fluorescent protein (VSV-GFP) was generously provided by Prof. Zhigao Bu from the Harbin Veterinary Research Institute, China. Rabbit polyclonal antibodies against p65, IRF3, phosphorylated IRF3 (p-IRF3), and DDX1 were purchased from ABclone (Wuhan, China). Rabbit polyclonal antibody against phosphorylated p65 (p-p65) was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin antibody was purchased from Beyotime (Nantong, China). Mouse monoclonal antibodies (MAbs) against hemagglutinin (HA) and Flag were purchased from Medical and Biological Laboratories (MBL, Nagoya, Japan). MAb against TGEV N protein was prepared by our laboratory. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody and HRP-conjugated goat anti-mouse antibody were purchased from MBL. Alexa Fluor 594-conjugated donkey anti-rabbit IgG, Alexa Fluor 594-conjugated donkey anti-mouse IgG, and Alexa Fluor 488-conjugated donkey anti-mouse IgG were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).