rabbit polyclonal anti cyc1  (Proteintech)

Journal: eLife

Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

doi: 10.7554/eLife.67624

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Article Snippet: Antibody , Rabbit polyclonal anti-CYC1 , Proteintech , 10242–1-AP , RRID: AB_2090144 (1:1000).

Techniques: Control, Transduction, shRNA, Knockdown, Software

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 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. 2 Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 4 (nsp4) induces proteolytic cleavage of cytochrome c1 (cyto.c1). A, dose-dependent response to cyto.c1 cleavage by PRRSV nsp4. HEK 293T cells were transfected to express Flag-cyto.c1 together with increasing amount of HA-nsp4. At 24 h post-transfection, the cleavage of cyto.c1 was analyzed by Western blot with antibodies to Flag, HA, and β-actin. B, the nsp4 protease activity is required for cleaving cyto.c1. The same as A, except that HEK 293T cells were transfected to express Flag-cyto.c1 with nsp4 mutants. C, the nsp4 protease activity was required for activate caspase-3. The same as A, except that MARC-145 cells were used. D, nsp2 does not cleave cyto.c1. HEK 293T cells were transfected to express Flag-cyto.c1 together with nsp2. E, identification of nsp4 cleavage site in cyto.c1. The same as A, except that HEK 293T cells were transfected to express HA-nsp4 together with cyto.c1 mutants. F, size comparison of cyto.c1 (aa 1–230) with the cyto.c1 cleaved fragment. HEK293 T cells were transfected to express Flag-cyto.c1 (aa 1–230) or co-express nsp4 and cyto.c1. G, nsp4-induced cleavage does not depend on caspase-3 activation. Transfected HEK293 T cells expressing nsp4 and cyto.c1 were either treated with Z-VAD-FMK or untreated (left). Meanwhile, the untransfected cells were treated with STS at 6 h before cells were harvested. At 30 h post transfection, the cells were analyzed by immunoblotting with indicated antibodies. H, PRRSV HB-1/3.9 nsp4 induces cleavage of cyto.c1. HEK293 T cells were transfected to co-express cyto.c1 and nsp4 from PRRSV strain HB-1/3.9, and the cleavage was analyzed by Western blot analysis of the whole cell lysate.

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, Transfection, Western Blot, Activity Assay, Comparison, Activation Assay, Expressing

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. 3 Cytochrome c1 (cyto.c1) is cleaved during porcine reproductive and respiratory syndrome virus (PRRSV) infection. MARC- 145 cells were either mock-infected with Dulbecco’s Modified Eagle medium (DMEM) or infected with highly pathogenic (HP)-PRRSV at a multiplicity of infection (MOI) of 0.1 or 1. At different times after infection as indicated, the cells were harvested and subjected to Western blot analysis with antibodies to caspase-3, cyto.c1, nsp4 and β-actin.

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, Infection, Modification, Western Blot

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. 4 The cleaved N-terminal fragment of cytochrome c1 (cyto.c1) induces cell apoptosis. MARC-145 cells were transfected to express Flag-cyto.c1 or its derivatives as indicated. At 48 h post transfection, the cells were harvested and subjected to SDS- PAGE and Western blot analysis with antibodies to caspase-3, Flag and β-actin, respectively.

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: Transfection, SDS Page, Western Blot

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. 5 Cytochrome c1 (cyto.c1) (aa 1–230) induces mitochondrial fragmentation of mammalian cells. Vero (A), HeLa (B) or MARC- 145 (C) cells grown on coverslips in six-well plates were transfected when 60–70% to express Flag-cyto.c1 and its derivatives. At 18–24 h post-transfection, the cells were first stained with mitotracker, and then fixed, permeablized, and stained with antibodies to Flag followed by Alex-488 conjugated goat anti-mouse IgG Fab fragment. Images were analysed with a Nikon confocal microscope (Nikon Instruments Inc., Tokyo, Japan), and representative images were shown. Bar=10 µm.

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: Transfection, Staining, Microscopy

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. 6 Cytochrome c1 (cyto.c1) is critical for porcine reproductive and respiratory syndrome virus (PRRSV)- and nonstructural protein 4 (nsp4)-induced cell apoptosis. A, MARC- 145 cells were transfected with either siRNAs targeting cyto.c1 or scrambled siRNA (siRNA-NC). At 48 h post transfection, the cells were subject to SDS-PAGE and analyzed with immunoblotting with antibodies to cyto.c1 and β-actin. B, MARC-145 cells were first transfected with either siRNA-NC or siRNA-3. At 24 h post transfection, the cells were infected with HP-PRRSV strain JXwn06 at an MOI of 0.1. At 48 h post infection, the cells were harvested and subject to either FACS analysis with PI or FITC-conjugated annexin V. C, the same as B, except that the cells were used for western blot analysis with antibodies to caspase-3, nsp4 and β-actin. D, the same as B, except that the cells were transduced with the lentivirus expressing nsp4-GFP. The experiments are representative of three independent repeats.

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, Transfection, SDS Page, Western Blot, Infection, Transduction, Expressing