Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 1 AChE interacts with RanBPM (A) The self-expression assay of the bait. AH109/pGBKT7-AChEC-terminal cells were cultured on SD/-Trp plate or SD/-Trp/-His plates. (B) Library screening. AH109 clones transfected with pGBKT7-AChEC-terminal and human fetal brain library were cultured on SD/-Ade/-His/-Leu/-Trp/X-a-gal plates. The arrows indicate the positive clones (blue clones). (C) The scheme of the two positive clones (A and C6). The putative SPRY domain (amino acids 212–333) is indicated. (D) Clones A and C6 showed strong lacZ reporter activation. Pos., a clone consisting of BD-p53 and AD-SV40 T-antigen was used as the positive control. Neg., a combination of BD-laminC and AD-SV40 T-antigen was used as the negative control; b-galactosidase activity is expressed as a percentage of the positive control. The data are represented as the mean+ SD of three independent transformations. (E,F) HEK293T cells transfected with the indicated expression plasmid for 24 h; the whole-cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting with anti-HA antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-HA (middle panel) or anti-Myc (bottom panel) to confirm the expression of HA-RanBPM or Myc-AChEC-terminal (or Myc-AChE), respectively. (G) Kidney tissues from the rat model of ischemia/reperfusion injury were immunoprecipitated using the anti-AChE antibody (E70), and analyzed by immunoblotting with the anti-RanBPM antibody (top panel). Separate aliquots of the lysates were immunoblotted with anti-RanBPM (middle panel) or anti-AChE (E70) (bottom panel) to confirm the expression of RanBPM or AChE, respectively, in the rat kidney tissues.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Expressing, Cell Culture, Library Screening, Clone Assay, Transfection, Activation Assay, Positive Control, Negative Control, Activity Assay, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 3 Subcellular distribution of RanBPM and AChE (A,B) Fractionation of HEK293T cell extracts. HEK293T cells were transfected with the indicated expression plasmids. At 24 h after transfection, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions and analyzed directly by immunoblotting with the anti-HA (first panel) or anti-Myc antibody (second panel). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-histone 3 (fourth panel) and anti-a-tubulin antibody (third panel), respectively. (C) Determination of co-localization of AChE and RanBPM by immunofluorescence. HEK293T cells were transfected with Myc-RanBPM and GFP-AChE. At 24 h after transfection, the cells were fixed and incubated with anti-Myc (red), followed by incubation with Rhodamine-conjugated secondary antibodies. The cells were examined by fluorescence microscopy.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Fractionation, Transfection, Expressing, Western Blot, Incubation, Fluorescence, Microscopy

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 4 Subcellular distribution of RanBPM and AChE after cisplatin treatment (A) HEK293T cell viability was assessed by MTT after treatment with different concentrations of cisplatin for 24 h. The graph shows the mean+ SE of three independent experiments. (B) The transcription level of p73a gene detected by RT–PCR. HEK293T cells were treated with different concentrations of cisplatin for 24 h and then total mRNA was extracted for RT–PCR. (C) Cisplatin of 100 mM or higher induced DNA fragmentation. (D) The amount of RanBPM and AChE in the nuclear fractions increased after treatment with cisplatin. After cotransfection with pCMV-Myc-RanBPM and pEGFP-C1-AChE for 24 h, HEK293T cells were treated with the indicated concentrations of cisplatin for another 24 h. Then, the cells were fractionated into nuclear (N) and cytoplasmic (C) fractions. The cell fractions and the whole-cell lysates were immunoblotted with anti-Myc (left line) or anti-GFP (light line). The graph shows the densitometric values of the proteins in the cytoplasm versus those in the nuclei.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cotransfection

Journal: Acta biochimica et biophysica Sinica

Article Title: RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis.

doi: 10.1093/abbs/gmp082

Figure Lengend Snippet: Figure 5 Nuclear distribution of endogenous RanBPM and AChE after long-term culture (A) Cleaved caspase 3 was detected in long-term cultured HEK293T cells. HEK293T cells were cultured without medium changed for 3 days (3D) or 7 days (7D), and then the whole-cell lysates were immunoblotted with anti-capspase 3, or anti-b-actin antibody. (B) The amount of endogenous RanBPM and AChE in the nuclear fractions was increased after long-term culture. HEK293T cells were cultured without medium changed for 2 days (2D), 5 days (5D) or 8 days (8D), and then the nuclear fractions were immunoblotted with anti-AChE (first panel) or anti-RanBPM antibody (second panel, NB 100–1281). The purity of the nuclear and cytoplasmic fractions was examined by immunoblotting with anti-a-tubulin (third panel) or anti-histone 3 antibody (fourth panel), respectively.

Article Snippet: The primary antibodies used were as follows: anti-RanBPM antibody (NB100-1281; Novus Biologicals) (1:1000), anti-AChE antibody (the antibody against AChE was prepared in the rabbit [37]) (1:1000), anti-Myc antibody (1:5000), anti-AChE antibody (1:1000), anti-GFP antibody (sc-9996; Santa Cruz) (1:1000), anti-HA antibody (sc-805; Santa Cruz) (1:500), anti-caspase 3 antibody (Cell Signalling, #9662) (1:500), anti-histone 3 antibody (ab1791; ABCAM) (1:3000), anti-tubulin antibody (T6074; Sigma) (1:5000), anti-actin antibody (A5441; Sigma) (1:5000).

Techniques: Cell Culture, 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. 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 molecular medicine

Article Title: Role of angiotensin II type 1 receptor in cerebral aneurysm formation in rats.

doi: 10.3892/ijmm_00000239

Figure Lengend Snippet: Figure 5. Immunostaining of NF-κB, MCP-1, MMP-2 and MMP-9 in cerebral aneurysmal walls and zymography of MMP-2 and MMP-9 activities. Immunostaining of DNA binding form of NF-κB p65 subunit (C and D), MCP-1 (E and F), MMP-2 (G and H) and MMP-9 (I and J) in cerebral aneurysmal walls of rats with vehicle (C, E, G, I) or valsartan treatment (D, F, H, J). (A and B) Elastica van Gieson staining (EvG) of serial section as C (A) or D (B). Bar: 30 μm. (K) Representative image of gelatine zymography from five independent experiments.

Article Snippet: Primary antibodies used in this study are listed below: rabbit polyclonal anti-monocyte chemoattractant protein (MCP-1) antibody (Santa Cruz, Santa Cruz, CA), mouse monoclonal anti-smooth muscle ·-actin antibody (Lab Vision, Fermont, CA), goat polyclonal anti-CD68 antibody (Santa Cruz), mouse monoclonal anti-NF-κB p65 subunit antibody which recognizes only DNA-binding form (Chemicon, Temecula, CA), rabbit polyclonal anti-matrix metalloproteinase (MMP)-2 antibody (Santa Cruz), goat polyclonal anti-MMP-9 antibody (Santa Cruz), rabbit polyclonal anti-AT1R antibody (Santa Cruz), and rabbit polyclonal anti-AT2R antibody (Santa Cruz).

Techniques: Immunostaining, Zymography, Binding Assay, Staining

Journal: International journal of molecular medicine

Article Title: Role of angiotensin II type 1 receptor in cerebral aneurysm formation in rats.

doi: 10.3892/ijmm_00000239

Figure Lengend Snippet: Figure 4. Effect of valsartan on MCP-1, MMP-2 and MMP-9 mRNA expression. (A-C) Result of quantitative real-time PCR analysis of MCP-1 (A), MMP-2 (B) and MMP-9 (C) with vehicle or valsartan treatment (n=6).

Article Snippet: Primary antibodies used in this study are listed below: rabbit polyclonal anti-monocyte chemoattractant protein (MCP-1) antibody (Santa Cruz, Santa Cruz, CA), mouse monoclonal anti-smooth muscle ·-actin antibody (Lab Vision, Fermont, CA), goat polyclonal anti-CD68 antibody (Santa Cruz), mouse monoclonal anti-NF-κB p65 subunit antibody which recognizes only DNA-binding form (Chemicon, Temecula, CA), rabbit polyclonal anti-matrix metalloproteinase (MMP)-2 antibody (Santa Cruz), goat polyclonal anti-MMP-9 antibody (Santa Cruz), rabbit polyclonal anti-AT1R antibody (Santa Cruz), and rabbit polyclonal anti-AT2R antibody (Santa Cruz).

Techniques: Expressing, Real-time Polymerase Chain Reaction