c jun inhibitor sr11302  (Tocris)

Journal: Frontiers in Microbiology

Article Title: The CREB and AP-1–Dependent Cell Communication Network Factor 1 Regulates Porcine Epidemic Diarrhea Virus-Induced Cell Apoptosis Inhibiting Virus Replication Through the p53 Pathway

doi: 10.3389/fmicb.2022.831852

Figure Lengend Snippet: The CREB binding site and AP-1 binding site are indispensable for CCN1 production. (A) Schematic diagram represents the –985/203-luc CCN1 promoter deletion mutant vectors. (B) The –985/203-luc CCN1 promoter and variety of deletion mutant were transfected into Marc-145 cells for 24 h. The cells were infected with PEDV for 48 h determining the luciferase activity. (C,D) Marc-145 cells were treatment using EML-425 (CREB inhibitor) with different concentrations at 1 h and then using PEDV incubated for 48 h. Real-time PCR was used to analyze CCN1 levels (C) , and Western blotting was used to analyze the protein expression of CCN1 (D) . (E) Densitometric analysis of CCN1 relative to β-actin using ImageJ. (F,G) Marc-145 cells were treatment using SR11302 (AP-1 inhibitor) at 1 h and then using PEDV incubated for 48 h. Real-time PCR was used to analyze CCN1 levels (F) , and Western blotting was used to analyze the protein expression of CCN1 (G) . (H) Densitometric analysis of CCN1 relative to β-actin using ImageJ. (I) Marc-145 cells were incubated with PEDV, and cells were collected at 12, 24, and 36 h. Western blotting was used to examine the level of CREB and phospho-CREB. (J) Densitometric analysis of phospho-CREB relative to CREB using ImageJ. (K) Marc-145 cells were infected with PEDV, cells were harvested at 48 h, and the mRNA level of c-Jun and c-Fos were analyzed using real-time PCR. (L) Marc-145 cells were infected with PEDV, and cells were harvested at 12, 24, and 36 h. Western blotting was used to examine the level of c-Jun and phospho-c-Jun. (M) Densitometric analysis of phospho-c-Jun relative to β-actin using ImageJ. (N) Marc-145 cells were infected with PEDV, and cells were harvested at 12, 24, and 36 h. Western blotting was used to examine the level of c-Fos and phospho-c-Fos. (O) Densitometric analysis of phospho-c-Fos relative to β-actin using ImageJ. The data were performed from three independent experiments. The differences were evaluated using Student’s t -test, and significance differences were denoted by * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: p38 inhibitor SB203580 (10 μM), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μM), extracellular signal-regulated kinase (ERK) inhibitor PD98059 (5 μM), PKA inhibitor H-89 (5 μM), c-Jun inhibitor SR11302 (5 μM), CREB inhibitor EML-425 (5 μM), and p53 inhibitor PFT-α (10 μM) are all purchased from MedChemExpress (Shanghai, China).

Techniques: Binding Assay, Mutagenesis, Transfection, Infection, Luciferase, Activity Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Expressing