Journal: Nature Communications

Article Title: Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis

doi: 10.1038/s41467-017-00834-8

Figure Lengend Snippet: Anti-inflammatory effects of CLT and CLT-AN in anti-Thy1.1 nephritic rats. a – d Real-time PCR analysis of renal mRNA levels of MCP-1 ( a ), ICAM-1 ( b ), IL-6 ( c ), IL-1β ( d ) in anti-Thy1.1 nephritic rats on day 1 after treatment with CLT or CLT-AN. Data are shown as normalized fold expressions relative to normal group using β-actin mRNA as internal control. e Representative photomicrographs of immunostaining for MCP-1, ICAM-1, IL-6, and IL-1β in kidney tissue sections taken from anti-Thy1.1 nephritic rats on day 1 after treatment with CLT or CLT-AN. Scale bars , 20 μm. f The levels of MCP-1, ICAM-1, IL-6, and IL-1β were semiquantitatively scored as described in Methods on the basis of immunohistochemical results. In panels a – d and f data are mean ± s.d. ( n = 5), results are representative of two independent experiments. * P < 0.05 vs. PBS group; # P < 0.05 vs. CLT group. Statistical significance was determined by one-way ANOVA with Tukey post hoc test

Article Snippet: After addition of 100 μL Agent A (horseradish peroxidase-conjugated ChemMate Envision reagent) from the anti-rabbit/rat general immunohistochemical test kit (Envision Detection Kit, GK500705), the color reaction was performed using 3,3-diaminobenzidine (Sigma-Aldrich, USA), then each section was counterstained with hematoxylin.

Techniques: Real-time Polymerase Chain Reaction, Control, Immunostaining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis

doi: 10.1038/s41467-017-00834-8

Figure Lengend Snippet: Anti-proliferative effects of CLT and CLT-AN in vitro and in vivo. a Effects of CLT and CLT-AN on PDGF-BB-induced proliferation of HBZY-1 cells. b Flow cytometry analysis of the effects of CLT and CLT-AN on the cell cycle distribution of HBZY-1 cells in the presence of PDGF-BB ( C CLT = 0.25 μg mL −1 ). c Representative quadrant plot obtained by flow cytometry analysis showing the ability of CLT and CLT-AN to induce apoptosis in HBZY-1 cells in the presence of PDGF-BB. d Flow cytometry analysis of the proportions of apoptotic HBZY-1 cells after 24-h exposure to CLT or CLT-AN in the presence of PDGF-BB ( C CLT = 0.5 μg mL −1 ). In panels a , b , and d data are mean ± s.d. ( n = 3), results are representative of three independent experiments. * P < 0.05. Statistical significance was determined by one-way ANOVA with Tukey post hoc test. e Real-time PCR analysis of renal mRNA levels of PDGF-BB in anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN. Data are shown as normalized fold expressions relative to normal group using β-actin mRNA as internal control. f Representative photomicrographs of immunostaining for PDGF-BB in kidney tissue sections from anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN, and the levels of PDGF-BB were semiquantitatively scored as described in Methods on the basis of immunochemical results. Scale bars , 20 μm. In panels e , f data are mean ± s.d. ( n = 5), results are representative of two independent experiments. * P < 0.05 vs. PBS group; # P < 0.05 vs. CLT group. Statistical significance was determined by one-way ANOVA with Tukey post hoc test

Article Snippet: After addition of 100 μL Agent A (horseradish peroxidase-conjugated ChemMate Envision reagent) from the anti-rabbit/rat general immunohistochemical test kit (Envision Detection Kit, GK500705), the color reaction was performed using 3,3-diaminobenzidine (Sigma-Aldrich, USA), then each section was counterstained with hematoxylin.

Techniques: In Vitro, In Vivo, Flow Cytometry, Real-time Polymerase Chain Reaction, Control, Immunostaining

Journal: Nature Communications

Article Title: Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis

doi: 10.1038/s41467-017-00834-8

Figure Lengend Snippet: Anti-fibrotic effects of CLT and CLT-AN in anti-Thy1.1 nephritic rats. a – d Real-time PCR analysis of renal mRNA levels of Col I ( a ), Col IV ( b ), FN-1 ( c ), TGF-β 1 ( d ) in anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN. Data are shown as normalized fold expressions relative to normal group using β-actin mRNA as internal control. e Representative photomicrographs of immunostaining for TGF-β 1 in kidney tissue sections taken from anti-Thy1.1 nephritic rats on day 5 after treatment with CLT or CLT-AN. Scale bars , 20 μm. f The levels of TGF-β 1 were semiquantitatively scored as described in Methods on the basis of immunochemical results. In panels a – d , f data are mean ± s.d. ( n = 5), results are representative of two independent experiments. * P < 0.05 vs. PBS group; # P < 0.05 vs. CLT group. Statistical significance was determined by one-way ANOVA with Tukey post hoc test

Article Snippet: After addition of 100 μL Agent A (horseradish peroxidase-conjugated ChemMate Envision reagent) from the anti-rabbit/rat general immunohistochemical test kit (Envision Detection Kit, GK500705), the color reaction was performed using 3,3-diaminobenzidine (Sigma-Aldrich, USA), then each section was counterstained with hematoxylin.

Techniques: Real-time Polymerase Chain Reaction, Control, Immunostaining

Journal: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Rasip1 expression in IAHCs in the E10.5 AGM region. A . Upper panel: The expression profile of CD45, c-Kit, CD31, and VE-Cad in the E10.5 AGM region. The numbers from 1 to 4 indicate cell populations separated by the expression profile: 1.CD45 − c-Kit −- CD31 + VE-cad − cells (endothelial cells); 2.CD45 − c-Kit − CD31 + VE-cad + cells (endothelial cells); 3.CD45 low c-Kit high cells (containing HSPCs); 4.CD45 + c-Kit − cells (hematopoietic cells). Lower panel: RT-PCR shows the relative expression levels of Rasip1 in these 4 populations. B . Whole-mount immunofluorescence staining of Rasip1 (green), CD31 (red), and c-Kit (cyan) in IAHCs of the E10.5 AGM region. The Rasip1 expression on the membrane of IAHCs. The nuclei were stained by Hoechst 33258 (blue). Scale bar, 20 μm. C . A genetic scheme showing the location of the Rasip1 gene and upstream genes in mouse chromosome 7. 5 putative Sox17-binding sites were indicated by squares with numbers 1 through 5. D . Upper panel: Requirement of the proximal putative Sox17-binding site (5) for induction of the Rasip1 gene by Sox17. NIH3T3 cells (1 × 10 6 ) were transfected with a pGL3 vector containing the putative Rasip1 promoter sequences. pRL-CMV was co- transfected as an internal control. The region Rasip1 [ - ] (979 bp) has three putative Sox17- binding sites (1, 2, and 3), and the region Rasip1 [ - ] (602 bp) has two putative Sox17 binding sites (2 and 3). The region Rasip1 [ - ] (720 bp) has putative Sox17-binding sites 4 and 5. Two types of Sox17-binding sites are shown in the promoter regions: 1. AATGGCG (Sox17 binding sites 2, 4); 2. ATTGT (Sox17 binding sites 1,3,5). Lower panel: Inhibition of the Sox17-induced Rasip1 gene activation by a mutation in the putative Sox17-binding site 5. NIH3T3 cells (1 × 10 6 ) were transfected with a pGL3 vector containing the putative Rasip1 promoter sequences with point mutations in the putative Sox17-binding sites 4 or 5, or both together with plasmids encoding wild-type (WT) Sox17 or Sox17G103R which lost the DNA binding ability. In this experiment, pRL-TK was co-transfected as an internal control. A solid black line indicates the Rasip1 [ - ] region, solid grey lines show the pGL3 vector and Rasip1 promoter region [ - ] as controls.

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Binding Assay, Transfection, Plasmid Preparation, Inhibition, Activation Assay, Mutagenesis

Journal: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Expression of Rasip1 in Sox17 (GFP)-transduced CD45 low c-Kit high cells. A . Upper panel: A schema of Sox17-IRES-GFP (Sox17) and IRES-GFP (Mock) retrovirus vectors. Lower panel: The red squares show a region of the CD45 low c-Kit high cell population. The expression of Rasip1 in Mock- or Sox17-transduced cells was analyzed by RT-PCR and normalized by β-actin. B . Upper panel: A schema of the Sox17-ERT-IRES-GFP retrovirus vector. Lower panel: The dark green squares show Sox17 (GFP) + CD45 low c-Kit high cells. After FACS sorting, expression analysis of the Rasip1 gene was performed in Sox17-ERT- IRES-GFP-transduced cells with or without tamoxifen (1.0 μg/ml).

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Rasip1 knockdown (KD) in CD45 low c-Kit high cells. A . Upper panel: A schema of the position of the Rasip1 shRNA in the Rasip1 mRNA, a retrovirus vector of Sox17-IRES- mCherry, and retrovirus vectors of shLuc and shRasip1. Lower panel: After 4 days of Sox17- IRES-mCherry transduction, Sox17-transduced cells formed cell clusters. shLuc and shRasip1 were introduced into Sox17-transduced cells and the cells were co-cultured with OP9 stromal cells. Morphologies of shRasip1 (green, GFP + )-transduced Sox17 + cells (red, mCherry + ). Scale bar, 1.0 mm. B . shLuc- or shRasip1-transduced GFP + cells were recovered by FACS. Expression of Rasip1 in shLuc- and shRasip1-transduced cells was analyzed by RT-PCR and normalized by β-actin gene expression. RT-PCR result is in the static image that is generated by a mirror-reversal of an original across a horizontal axis, due to the sample order in the electrophoresis. C . Sorted GFP + cells (2.5 × 10 3 ) were embedded in a semi-solid medium. The number of total (CFU-C) and mix (CFU-Mix) colonies were scored after 7 days of culture. The pair of colony numbers (sh-Luc and shRasip1) in each experimental group were plotted and connected by a line. The different marks indicate the different individual embryos (n = 7, 7 individual liters of mice from which each experimental group was set up).*p≤0.05; **p≤0.01; ***p≤0.005.

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: shRNA, Plasmid Preparation, Transduction, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Electrophoresis

Journal: bioRxiv

Article Title: A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

doi: 10.1101/2022.11.01.513960

Figure Lengend Snippet: Rasip1 overexpression (OE) in CD45 low c-Kit high cells. A . A schema of IRES-GFP (Mock) and Rasip1-IRES-GFP retrovirus vectors. B . Timeline of Rasip1 OE experiment. C . Red squares show Mock- and Rasip1-transduced CD45 low c-Kit high cells. D . After 4 days of Mock and Rasip1 transduction, CD45 low c-Kit high cells (2.5 × 10 2 ) were embedded in a semi- solid medium (Colony assay 1). According to the timeline in , CD45 low c-Kit high cells were sorted (5.0 × 10 2 ) after 8 days of Rasip1 transduction (Colony assay 2). The number of total (CFU-C) and multilineage (CFU-Mix) colonies were scored after 7 days of culture (n=5). The pair of colony numbers (Mock and Rasip1) in each experimental group were plotted and connected by a line. The different marks indicate the different individual embryos (n=6, 6 individual liters of mice from which each experimental group was set up). *p≤0.05; **p≤0.01.

Article Snippet: After washing with PBS, the embryos were treated with PBS-MT [1% (w/v) skim milk powder, and 0.4% (v/v) Triton X-100] containing 2% (w/v) bovine serum albumin for 1 h on ice and then immunostained with a rat anti-mouse CD117 (c-Kit) antibody (2B8; BD Pharmingen), a goat anti-mouse CD31 antibody (Bio-Techne, Minneapolis, MN, USA), a rabbit anti-mouse Rasip1 antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Over Expression, Transduction, Colony Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MTSS1/Src family kinase dysregulation underlies multiple inherited ataxias

doi: 10.1073/pnas.1816177115

Figure Lengend Snippet: MIMEX15-mutant Purkinje neurons undergo autophagy. (A) MIMEX15 mutants display fused mitochondria shown by increased complex 5 ATP-synthase immunostaining and collapsed Golgi shown by reduced giantin immunostaining at 4 wk. (B) Eight-week-old MIMEX15 mutants show increased LC3-II abundance. *P < 0.005; Student’s t test. (C) MIMEX15 mutants show increased levels of mRNA for the autophagocytic marker VMP1. *P < 0.05; Student’s t test. (D) MIMEX15 mutants show increased microglial infiltration shown by Aif1 transcript. *P < 0.002; Student’s t test. (E) MIMEX15 mutants show GFAP+ glial infiltration during disease progression. (F) Western blots quantifying increased cerebellar GFAP. *P < 0.05; Student’s t test. (G) MIMEX15-mutant cerebella do not have increased TUNEL staining at 4, 8, or 16 wk of age.

Article Snippet: The following antibodies were used at 1:1,000 dilutions: rabbit anti-Mtss1 ( 30 ), rabbit anti-calbindin (13176; Cell Signaling Technology ), mouse anti–calbindin-D-28K monoclonal (Sigma), mouse anti-complex V (Novex; 459240), rabbit anti-ubiquitin (3933; Cell Signaling Technology), rabbit anti-giantin (Abcam; ab24586), and chicken anti-GFAP (ab4674; Abcam).

Techniques: Mutagenesis, Immunostaining, Marker, Western Blot, TUNEL Assay, Staining

Journal: Oncogene

Article Title: Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth

doi: 10.1038/s41388-020-01523-5

Figure Lengend Snippet: A Western blotting showing the expression of p-STAT3 and STAT3 in healthy colon or colon tumors. B Densitometry quantification of p-STAT3/total STAT3 for colons or colorectal tumors. *** p < 0.001 (one-way ANOVA). C Representative images of p-STAT3 immunostaining of colons or colon tumors. Scale bar: 50 μm. D Quantification of p-STAT3 + cells in colon tumors of WT and Gal2-KO mice. *** p < 0.001 ( t -test).

Article Snippet: Primary antibodies including the goat polyclonal anti-Gal2 (STJ24400, 1:500, St John’s Laboratory Ltd, London, UK), anti-STAT3 (#9139, 1:1000), anti-p-STAT3 (#9145, 1:1000), and anti-GAPDH (MAB374, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Immunostaining

Journal: Oncogene

Article Title: Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth

doi: 10.1038/s41388-020-01523-5

Figure Lengend Snippet: A Western blotting showing the expression of Gal2 in stable Gal2-overexpressing HCT116 cells. The red arrow indicates the un-cleaved GFP-2A-Gal2 fusion protein. B The CCK8 proliferation assay of HCT116 with or without Gal2 overexpression. C The survival assay of HCT116 with or without Gal2 overexpression in the presence of 0.6 mM H 2 O 2 . ** p < 0.01, *** p < 0.001 ( t -test). D Western blotting showing the expression of p-STAT3 and STAT3 in HCT116 cells with or without Gal2 overexpression. E Densitometry quantification of p-STAT3/total STAT3 in HCT116 cells with or without Gal2 overexpression. ** p < 0.01 (one-way ANOVA).

Article Snippet: Primary antibodies including the goat polyclonal anti-Gal2 (STJ24400, 1:500, St John’s Laboratory Ltd, London, UK), anti-STAT3 (#9139, 1:1000), anti-p-STAT3 (#9145, 1:1000), and anti-GAPDH (MAB374, 1:2000) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Expressing, Proliferation Assay, Over Expression, Clonogenic Cell Survival Assay

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: Confocal sections from retinas of E6 chick embryos were double labeled with the anti-cdk1 specific mAbs (red) (B-6) ( A ), [A17] ( B ), and (17) ( C - E ), as well as with anti-p75 NTR (p75) ( C ), anti-phosphoHistone H3 (pH3) ( D ), or anti-TrkB ( E ) antibodies (green). Nuclear staining with bisbenzimide is shown in blue (Bisb.). ( A , B ) Cdk1 immunostaining is observed in the cytoplasm of a subpopulation of retinal cells and often in nuclei located apically (arrows). ( C ) A subset of cdk1-positive cells co-localize with p75 NTR (arrowhead), whereas many other p75 NTR -positive cells lack cdk1 immunolabeling (arrow). ( D ) Cdk1-positive cells co-localize with phosphoHistone H3 in the basal retina, close to the presumptive GCL (arrow). Arrowhead: apically located nucleus with cdk1-specific immunoreactivity. ( E ) TrkB-positive cells co-localize with cdk1 (arrow). Boxes: high magnification of the dashed areas. GCL: ganglion cell layer; PE: pigment epithelium; v: vitreous body. Bar: 20 µm ( A - D ), 40 µm ( E ).

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Labeling, Staining, Immunostaining, Immunolabeling

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) Lysates from DCRNs cultured for 20 h in the presence of different combinations of 100 ng/m NGF and 2 ng/ml BDNF were subjected to western blot with antibodies specific for either cdk1 (upper panel) or β-actin (lower panel). ( B ) Normalized cdk1/β-actin ratio. ( C ) Semiquantitative RT-PCR analysis of mRNA samples obtained from DCRNs cultured for 20 h in the presence of either vehicle (Control) or 2 ng/ml BDNF (BDNF). The levels of Cdk1 expression were normalized to Gapdh . N.S. non-significant; *p<0.05 (Student’s t test; n = 3–4).

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) General cdk protein kinase activity, based on the phosphorylation of a Rb-derived peptide by the cdk activity present in cell extracts from DCRNs cultured for 20 h in the presence of 100 ng/ml NGF and treated for 30 min with either vehicle (Control) or 2 ng/ml BDNF. Equal amount of cell extracts were used in both cases. ( B ) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts described above prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts described above, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05 (ANOVA; n = 3).

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Activity Assay, Derivative Assay, Cell Culture, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) E6 retinal cells electroporated with either EGFP (-) or cdk1 plus cyclin B1 (CC) and EGFP (+) were cultured under neurogenic conditions for 20 h in the presence of different combinations of 100 ng/ml NGF and 2 ng/ml BDNF. The percentage of mitotic figures was evaluated in the EGFP-positive cells. Lower panels show an example of a mitotic figure (pH3; red) in an EGFP-transfected cell (EGFP) (arrow). ( B ) E6 retinal cells were electroporated and cultured as above. The percentage of pyknotic nuclei was evaluated in the EGFP-positive cells. Lower panels show an example of a pyknotic nucleus in an EGFP-transfected cell (EGFP) (arrow). Bisb.: bisbenzimide. *p<0.05; ***p<0.005 (Student’s t test; n = 4). Bars: 10 µm.

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Cell Culture, Transfection

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) Validation of the cdk1 sandwich ELISA assay performed with 10 fg of either GST (GST) or a chimeric protein containing the sequence of human cdk1 bound to GST (Cdk1-GST), and revealed with the anti-cdk1 PSTAIRE antibody. Normalized optical density (O.D.) at 405 nm is shown. ( B ) Validation of the phospho-cdk1 sandwich ELISA assay performed with cell lysates obtained from DCRNs cultured in the presence of 100 ng/ml NGF and 2 ng/ml BDNF, incubated either in the absence (- CIP) or presence (+ CIP) of CIP, and revealed with the anti-cdk1 (pTyr15) antibody. Normalized optical density (O.D.) at 405 nm is shown. ( C ) Cell lysates from DCRNs cultured in the presence (+) or absence (-) of the referred factors were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( D ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or K252a were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). *p<0.05; ***p<0.005 (Student’s t test; n = 3).

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Sandwich ELISA, Sequencing, Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: ( A ) Cell lysates from DCRNs cultured in the presence of the referred factors and/or 300 nM MK-1775 were subjected to sandwich ELISA analyses using antibodies specific for either cdk1 phosphorylated at Tyr15 or total cdk1 protein. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ( B ) Cell lysates from CEFs cultured in the presence (+) or absence (-) of 300 nM MK-1775 were subjected to sandwich ELISA analyses as described above. Colorimetric values for cdk1 phosphorylated at Tyr15 were normalized to the levels of cdk1 protein (Cdk1). ***p<0.005 (Student’s t test; n = 3). # p<0.05 (NGF/BDNF/MK-1775 vs NGF/MK-1775; Student’s t test; n = 3).

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Cell Culture, Sandwich ELISA

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: E6 retinal cells electroporated with either EGFP (-) or the Tyr15Phe mutant form of cdk1 plus cyclin B1 (Mut CC) and EGFP (+) were cultured for 20 h under neurogenic conditions in the presence of different combinations of 100 ng/ml NGF and 2 ng/m BDNF. The percentage of pyknotic nuclei was evaluated in EGFP-positive cells. *p<0.05 (Student’s t test; n = 4).

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Mutagenesis, Cell Culture

Journal: PLoS ONE

Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons

doi: 10.1371/journal.pone.0064890

Figure Lengend Snippet: TrkB activation by BDNF prevents the increase cyclin B and cdk1 protein expression (dashed line) and induces phosphorylation of cdk1 at Tyr15 (thick black arrow), thus inhibiting cdk1 kinase activity. This effect participates in the G2/M arrest (grey line) observed in tetraploid RGCs.

Article Snippet: The rabbit anti-phospho-Rb (Ser780) antibody (HTScan CDK1/CycB Kinase Assay Kit; Cell Signaling Technology) was used at 1/1,000 dilution for sandwich ELISA.

Techniques: Activation Assay, Expressing, Activity Assay