Alexa fluor 546 goat anti mouse igg h l

The cells of all groups were, respectively, fixed with 4% paraformaldehyde and this was followed by a treatment with 0.1% Triton-X100 for 10 min. The cells were closed with 5% BSA at room temperature for 2 h and were then washed three times with PBST. The cells were incubated with an anti-PRRSV N mouse monoclonal antibody for 1 h. The cells were washed three times with PBST and incubated with Alexa FluorTM 546 goat anti-mouse IgG (H + L) (Invitrogen, NY, USA) for 1 h in the dark, and then dyed for 10 min with DAPI. Finally, these images were observed and taken by the fluorescence microscopy.

PAMs in the mock group and those individually infected with PRRSV or PCV2 were fixed with 4% paraformaldehyde followed by treatment with 0.1% Triton-X100 for 10 min. The PAMs were closed with 5% BSA for 2 h, then washed three times with PBST. Subsequently, the PAMs were incubated with PRRSV N protein-specific monoclonal antibody (Zoonogen, Beijing, China) or PCV2 Cap protein-specific monoclonal antibody (gifted by Qianyue Jin, Henan Academy of Agricultural Sciences) for 1 h, washed three times with PBST, incubated with fluorescein goat anti-mouse IgG(H + L) or Alexa Fluor TM 546 goat anti-mouse IgG (H + L) (Invitrogen, Waltham, MA, USA) for 1 h and then dyed for 10 min with DAPI. Finally, this was observed and these images were taken by fluorescence microscopy.

Antibodies against Caspase-3 (No. 9662), PARP (No. 9532), Pro-caspase 9 (No. 9508), Cleaved Caspase-9 (No. 7237), Cytochrome C (No. 4272), Bcl-2 (No. 2870), Bax (No. 5023) and PCNA (No. 13110) were from Cell Signaling Technology (CST), USA. Anti-maternal embryonic leucine zipper kinase (MELK) (No. ab155767) was obtained from Abcam, UK. Anti-serine-threonine kinase receptor-associated protein (STRAP) (No. sc-377,345) and Caspase 8 (No. sc-56,070) antibody was purchased from Santa Cruz Biotechnology, USA. Anti-phospho-MELK (Thr167, Ser171) (No. WG-00203P) and Anti-phospho- STRAP (Thr175, Ser179) (No. WG-00204P) were ordered from ABclonal Technology, China. Alexa fluor^® 488 goat anti-rabbit IgG(H + L) (No. CA11008s) and Alexa fluor^®546 goat anti-mouse IgG(H + L) (No. A11003) were obtained from Molecular Probes (Invitrogen). Beta-actin antibody (No. E021020–01) was from EarthOx, LLC, San Francisco, USA. Sanguinarine was purchased from National Institutes of Food and Drug Control (Beijing, China). Sanguinarine was dissolved in DMSO (MP, France) and diluted in culture medium for each experiment. The final concentration of DMSO didn’t exceed 0.1%.