Fig. 8.
Expression and/or phosphorylation of several cell cycle checkpoint proteins in PRRSV-infected MARC-145 cells. a PRRSV infection markedly induced the expression of p53, p-p53, 14–3-3σ, and p21 in MARC-145 cells. Cell lysates were prepared, and the expression of p53, p-p53, 14–3-3σ, and p21 was determined with western blot. MARC-145 cells treated with 50 ng/mL Noco. for 16 h served as a positive control (left). Targeted protein expression levels were quantitatively analyzed and compared with GAPDH expression levels using of Image J (right). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. b p-p53(Ser15) expression in MARC-145 cells was visualized using IFA. PRRSV- and mock-infected cells were stained for p-p53(Ser15) (red), F-actin (green), and DNA (blue) with p-p53(Ser15) antibody, Phalloidin, and DAPI stain at 48 h post-infection. Then, the cells were visualized using Leica microsystems (Leica AF6000, Germany) (× 630). c Interactions between p21 and Cdc2-cyclinB1 in MARC-145 cells induced by PRRSV infection. Dynabeads-Ab complex was prepared by incubating Cdc2 mouse mAb with Protein G Dynabeads using a Catch and Release(version 2.0) reversible immunoprecipitation system (ThermoFisher, USA). Then, the supernatants of mock-infected, PRRSV-infected, or nocodazole-treated cells lysis were added to the tubes containing Dynabeads-Ab complex and incubated overnight at 4 °C. After washing with PBS, p21Walf1/Cip1 rabbit mAb and cyclinB1 antibody were used to detect the Dynabeads®-Ab-Ag complex with western blot