Journal: mBio
Article Title: Porcine Reproductive and Respiratory Syndrome Virus Adapts Antiviral Innate Immunity via Manipulating MALT1
doi: 10.1128/mbio.00664-22
Figure Lengend Snippet: MALT1 expression is elevated upon PRRSV infection. (A and B) Marc-145 cells were infected with PRRSV HuN4 (MOI, 1) for the indicated time periods. (A) MALT1 mRNA and protein expression was detected in qRT-PCR and Western blot, respectively. (B) PRRSV N protein and MALT1 were immunostained with anti-PRRSV N MAb VH13 (green) and anti-MALT1 pAb (red), respectively. Nuclei were labeled with DAPI (blue). (C) Marc-145 cells were transfected with plasmids expressing different nsps or pCDNA3.1 empty vector (EV). mRNA levels of MALT1 were detected by qRT-PCR 24 h after transfection. (D) Marc-145 cells were transfected with nsp4, -7β, or -11 plasmids or EV. MALT1 protein levels were detected in Western blot analysis. (E) Marc-145 cells were cotransfected with His-nsp and MALT1 plasmids. nsp and MALT1 signaling was visualized using anti-His MAb (green) and anti-MALT1 MAb (red), respectively. (F) Marc-145 cells were incubated with different concentrations of IL-17 (10 to 500 ng/mL) for 12 h (left) or with 200 ng/mL of IL-17 for different time periods (right). MALT1 levels were detected in Western blot analysis. (G) Marc-145 cells were incubated with IL-17 inhibitor Y320 at different concentrations (0 to 300 nM) for 24 h and subsequently transfected with pCDNA-nsp11 or empty vector. Culture supernatant was replaced with complete medium containing Y320 at indicated concentrations at 6 h posttransfection. Cells were collected 24 h after transfection for detection of MALT1 expression in Western blot analysis.
Article Snippet: LPS and IL-17 (absin, Shanghai, China), MALT1 inhibitor Mi-2, lysosome inhibitor chloroquine (CQ) and IL-17 inhibitor Y320 (Selleck, China), and proteasome inhibitor MG-132 (Beyotime) were used at the designated concentrations, prepared in dimethyl sulfoxide (DMSO) (Sigma) or water according to the manufacturers’ instructions.
Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Labeling, Transfection, Plasmid Preparation, Incubation