Journal: Journal of Integrative Agriculture

Article Title: Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4

doi: 10.1016/s2095-3119(17)61670-8

Figure Lengend Snippet: Fig. 1 Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 4 (nsp4) interacts with cytochrome c1 (cyto.c1). A, yeast two hybrid assay. The plasmids pGBKT7-nsp4 and pGADT7-cyto.c1 were co-transformed into the yeast strain Y2HGold and selected on QDO/X/ABA plates. The top and second panels are the respective positive and negative controls. B, HEK 293T cells were transfected to express Flag-cyto.c1, or HA-nsp4 or together. At 24 h post-transfection, the cells were either subjected to direct Western blot analysis or lysed and immunoprecipitated with anti-HA antibodies. The proteins bound to sepharose beads were separated by SDS-PAGE, transferred to PVDF membranes, and probed with the antibodies to Flag and HA. C, the same as B except that nsp4 truncation mutants were used. D, MARC-145 cells were transduced with letiviruses that were expressing GFP or nsp4-GFP in the presence of 8 µg mL–1 polybrene, respectively. The cells were harvested 48 h post-transduction and cell lysates were immunoprecipitated using anti-GFP beads. The proteins bound to sepharose beads were subjected to Western blot analysis using an anti-cytochrome c1 polyclonal antibody or anti-GFP mAb. E, PRRSV nsp4 interacts with cyto.c1 in mammalian cells. Vero cells grown on coverslips in six-well plates were transfected when 60–70% to express the indicated proteins, either alone or pairwise. At 18–24 h post-transfection, the cells were fixed, permeablized, and stained with antibodies to HA and Flag, and examined by confocal microscopy. For double transfections, the merged images are shown at the right.

Article Snippet: Mouse anti-green fluorescent protein (GFP) mAb (66002-1-Ig) and rabbit anti-cytochrome c1 polyclonal antibodies (10242-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Virus, Y2H Assay, Transformation Assay, Transfection, Western Blot, Immunoprecipitation, SDS Page, Transduction, Expressing, Staining, Confocal Microscopy

Journal: International journal of cardiology

Article Title: Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia.

doi: 10.1016/j.ijcard.2013.11.033

Figure Lengend Snippet: Fig. 2. Isolation and identification of vascular endothelial cells from the aortic endothelium with collagenase. Representative photomicrographs (×400) of isolated cells treated histocytochemically with CD31 antibody and incubated with DiI-Ac-LDL. Cells with cytomembrane and cytoplasmic staining of amber color indicate vascular endothelial cells (black arrow), and cells with red fluorescence were identified as viable vascular endothelial cells (white arrow), which consisted of the majority of total treated cells, indicating a successful en- dothelial cell isolation from aortic endothelium.

Article Snippet: Then mouse polyclonal CD31 antibody (at 1:100 dilution; Boster Biological Technology, Inc, Wuhan, China) was applied and incubated at 37 °C for 1 h, then stained with SABC, enhanced by DAB, dehydrated in a graded ethanol series, and followed by dimethylbenzene treatment to improve transparency, and finally mounted with neutral gum.

Techniques: Isolation, Incubation, Staining, Cell Isolation