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: Journal of Virology

Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein

doi: 10.1128/JVI.01910-18

Figure Lengend Snippet: Nuclear localization of Rep from two TYLCV strains is controlled by the conserved lysines. (A) Subcellular localization of RFP-tagged RepTYLCV (TYLCV-Alb13) variants in N. benthamiana upon transient expression with Agrobacterium. Arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. Scale bars represent 5 μm. (B) Immunoblot of the Rep-RFP fusion proteins upon transient expression in N. benthamiana. Proteins were detected with an anti-RFP antibody. To demonstrate equal protein loading, the membranes were stained with Ponceau S. (C) Micrographs of epidermal leaf cells transiently expressing RepTYLCV K-to-A triple mutant-RFP and stained with ER-Tracker Green. From the left in order: RFP channel, ER-Tracker, merge of RFP and ER-Tracker, bright field, and graph representing the normalized fluorescence intensity of RFP and ER-Tracker (GFP) along the lines in the micrographs. Note that the two signals are shifted and do not overlap, indicating that they localize in different positions. Scale bars represent 50 μm. (D) Subcellular localization of RFP-tagged Rep TYLCV-Almeria WT and K-to-A variants in N. benthamiana. For each sample, one representative epidermal cell is shown with a 4× zoom of its nucleus; arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. The scale bars represent 5 μm. (E) Box plot showing the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel D; a total of 16 cells were analyzed. (F) Box plot depicting the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel A; a total of 8 cells per sample were analyzed. The statistical analysis used is described in the legend to Fig. 2.

Article Snippet: Immunodetection of the proteins was performed according to standard protocols using anti-RFP antibody (Chromotek 6G6; 1:1,000) to detect the Rep-RFP fusion proteins and antihemagglutinin (anti-HA) antibody (Roche 3F10; 1:2,000) for Rep-SCFP C fusions as primary antibodies and anti-rat (Pierce 31470; 1:10,000) or anti-mouse (Pierce 31430; 1:10,000) as secondary antibodies.

Techniques: Expressing, Fluorescence, Western Blot, Staining, Mutagenesis