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: Scientific Reports

Article Title: TDP43 nuclear export and neurodegeneration in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1038/s41598-018-22858-w

Figure Lengend Snippet: siRNA depletion of export proteins has limited effects on TDP43 localization. ( a ) The distribution of TDP43 WT -mApple, as estimated by the nuclear to cytoplasmic ratio (NCR) was measured 48 h after transient transfection with siRNA targeting nuclear exporters in primary rat cortical neurons. Statistical significance was calculated using 2-way ANOVA with Tukey’s adjustment. Error bars in ( a ) show mean ± s.d. ( b ) Automated analysis of subcellular protein distribution. Center panels demonstrate areas selected to measure average nuclear (within green ellipse) and cytoplasmic (between blue and red bounding lines) intensity. The effect of siRNA depletion on neuronal survival is plotted in ( c ). Depletion of XPO1, XPO7, or both XPO1 & 7 reduce the risk of death in transfected neurons by 20%, while NXF1 depletion more than doubles the risk of death. Cumulative hazard shown on the left, and instantaneous hazard on the right. Additional information, including the number of neurons (#), hazard ratio (HR), Cox proportional hazards p value (p), confidence interval (95% CI), and log rank p value (LR p) are shown for all experiments in ( d ). N = cells per condition (8-16 technical replicates per condition per experiment, minimum 3 experiments per condition). NCR was normalized to control for each biological replicate before pooling. ( e ) Summary of localization and survival effects. Black arrows indicate exporters chosen for further study.

Article Snippet: Detection was accomplished by incubating with anti-XPO7 primary antibodies (diluted 1:500 in blocking solution, (Proteintech, 12980-1-AP) overnight at 4 °C.

Techniques: Transfection, Control

Journal: Scientific Reports

Article Title: TDP43 nuclear export and neurodegeneration in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1038/s41598-018-22858-w

Figure Lengend Snippet: XPO1, XPO7 or NXF1 overexpression enhances cytoplasmic TDP43 localization. ( a , b ) Overexpression of untagged XPO7 in rodent primary neurons effectively reduces the nuclear to cytoplasmic ratio (NCR) of TDP43 WT -EGFP compared to cells transfected with empty vector (EV). ( c , d ) Overexpression of either XPO1 or NXF1 appended to the self-cleaving EGFP-2A peptide also results in significant reductions in TDP43 WT -mApple NCR, while XPO5 overexpression has no effect on TDP43 WT -mApple localization. Arrowheads indicate cytoplasmic TDP43 accumulation, while arrows highlight nuclear clearance of TDP43. ( e–g ) XPO7 overexpression has little effect on TDP43-dependent toxicity. Cumulative and instantaneous hazards shown in ( e ) and ( f ), respectively, and additional data, number of neurons (#), hazard ratio (HR), Cox proportional hazards p value (p), confidence interval (95% CI), and log rank p value (LR p) are indicated in ( g ). XPO1, XPO5 and NXF1 potentiate the toxicity of TDP43 WT -mApple overexpression ( h – j ). Data in a-j represent 3 biological replicates per condition, minimum 8 wells per replicate/condition. The NCR was normalized to control for each biological replicate before pooling, and plotted as mean normalized log (NCR) ± s.d. ****p < 0.0001, 2-way ANOVA with Tukey’s adjustment.

Article Snippet: Detection was accomplished by incubating with anti-XPO7 primary antibodies (diluted 1:500 in blocking solution, (Proteintech, 12980-1-AP) overnight at 4 °C.

Techniques: Over Expression, Transfection, Plasmid Preparation, Control

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 3. Compressive force up- regulated RANKL mRNA ex- pression in PDL cells. (A) RT- PCR analysis of PDL cells. PDL cells were loaded at different compressive forces (0, 0.5, 1.0, 2.0, or 3.0 g/cm2) for 24 h or at a constant compressive force (2 g/cm2) for 0, 0.5, 1.5, 6, 24, or 48 h. The results of one of four representative independent exper- iments are shown. (B) Densitom- etry analysis. The results are ex- pressed as the mean ratio to actin expression of four independent experiments.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 5. Exogenous PGE2 treat- ment increased RANKL mRNA expression in PDL cells. (A) RT- PCR analysis of PDL cells. PDL cells were stimulated with PGE2 (0, 109, 108, or 107 M) for 24 h. PDL cells were stimulated with PGE2 (107 M) for the indi- cated times. The results of one representative independent exper- iment out of four are shown. (B) Densitometry analysis. The re- sults are expressed as the mean ratio to actin expression of four independent experiments.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 6. The expression of RANKL protein increased with compres- sive force or PGE2 treatment. PDL cells were exposed to compressive force or PGE2 for 2 days or 4 days and then whole cell lysate from the cells was subjected to Western blot analysis with antibody against RANKL. Lanes 1 and 2 are controls. Lane 3 is the 2-day PGE2 treatment. Lane 4 is the 4-day PGE2 treatment. Lane 5 is the 2-day compressive force treatment. Lane 6 is the 4-day compressive force treatment. The results of one of three representative independent ex- periments are shown.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Western Blot

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 8. Indomethacin inhibited RANKL mRNA up-regulation stimulated by compressive force. (A) RT-PCR analysis of PDL cells. PDL cells were stimulated with compressive force with or without indomethacin. Lane a contains control PDL cells without indomethacin. Lane b contains compressive force-loaded PDL cells without indomethacin. Lane c contains control PDL cells with indomethacin. Lane d contains compressive force-loaded PDL cells with indo- methacin. The results of one of four representa- tive independent experiments are shown. (B) Densitometry analysis. The results are expressed as the mean ratio to actin expression of four independent experiments.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis.

doi: 10.1359/jbmr.2002.17.2.210

Figure Lengend Snippet: FIG. 9. A model illustrating the mechanism by which compressive force-loaded PDL cells induce osteoclastogenesis. First, compressive force induces COX-2 expression, promoting PGE2 production. Second, increasing PGE2 affects PDL cells via an autocrine mechanism, result- ing in the up-regulation of RANKL expression in PDL cells. RANKL expression in PDL cells was up-regulated by compressive force, whereas OPG expression in PDL cells was not affected by compressive force. Finally, up-regulated RANKL expression induces osteoclasto- genesis from osteoclast progenitors in compressive force-loaded PDL cells.

Article Snippet: The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (sc7627; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing

Journal: The British journal of nutrition

Article Title: Long-term resveratrol treatment prevents ovariectomy-induced osteopenia in rats without hyperplastic effects on the uterus.

doi: 10.1017/S0007114513003115

Figure Lengend Snippet: Fig. 5. Effect of resveratrol (Res) on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor kB ligand (RANKL). After treatment, femur samples were collected and the expressions of OPG and RANKL were analysed by immunohistochemical methods. ((a)–(f)) Representative photomicrographs of femur sections showing OPG expression in the different groups : (a) the ovariectomy (OVX) group; (b–d) the Res 20, 40 and 80 mg/kg groups; (e) the oestradiol replacement therapy (ERT) group; (f) the Sham group. ((g)–(l)) Representative photomicrographs of femur sections showing RANKL expression: (g) the OVX group; ((h)–(j)) the Res 20, 40 and 80 mg/kg groups; (k) the ERT group; (l) the Sham group. Images were acquired at 200£ magnification. Arrows indicate the OPG- or RANKL-positive cells. (A colour version of this figure can be found online at http://journals.cambridge.org/bjn).

Article Snippet: H. Zhao et al.838 B ri ti sh Jo u rn al o f N u tr it io n niversity Press of rat IL-6, TNF-a, OPG and RANKL primary antibodies (Boster Bio-Engineering Company, Limited).

Techniques: Immunohistochemical staining, Expressing