Structured Review

Merck KGaA atf4
CuET inhibits the p97 pathway and induces cellular unfolded protein response (UPR) a ) Proteasome inhibitor MG132-treated cells (5μM, 6h) accumulate both forms of NRF1 (120- and 110-KDa bands, upper and lower arrows, respectively) while CuET-treated cells (1μM, 6h) accumulate only the non-cleaved 120-KDa form; b ) Inhibition of the NRF1 cleavage process (appearance of the lower band) by CuET and NMS873 (a p97 inhibitor; 5μM) in mouse NIH3T3 cells co-treated with the proteasome inhibitor MG132 (5μM for 6h); c ) Time-course example images from a FRAP experiment the quantitative analysis of which is shown in Fig. 2g (U-2OS cells, blue boxes mark areas before bleaching, arrows after bleaching); d ) U-2OS cells pre-extracted by TritonX and stained for K-48-polyUb. The Ab signal intensities for cells treated with DMSO, BTZ (1μM), NMS873 (10μM) and CuET (1μM) are analysed by microscopy-based cytometry and plotted below; e ) Western blot analysis of accumulated poly-Ub proteins in ultracentrifugation-separated microsomal fraction from U-2OS cells treated by mock, CuET (1μM), NMS873 (10μM) or BTZ (1μM) for 3h; f ) UPR in U-2OS and MDA-MB-231 cell lines induced by 6-h treatment with CuET (various concentrations) or positive controls (NMS873 5μM, tunicamycin 2μg/ml, thapsigargin 1μM) manifested by increased levels of Xbp1s, <t>ATF4</t> and p-eIF2a. Panels a-f are representative of two independent experiments.
Atf4, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/atf4/product/Merck KGaA
Average 91 stars, based on 13 article reviews
Price from $9.99 to $1999.99
atf4 - by Bioz Stars, 2020-09
91/100 stars

Images

1) Product Images from "Alcohol-abuse drug disulfiram targets cancer via p97 segregase adapter NPL4"

Article Title: Alcohol-abuse drug disulfiram targets cancer via p97 segregase adapter NPL4

Journal: Nature

doi: 10.1038/nature25016

CuET inhibits the p97 pathway and induces cellular unfolded protein response (UPR) a ) Proteasome inhibitor MG132-treated cells (5μM, 6h) accumulate both forms of NRF1 (120- and 110-KDa bands, upper and lower arrows, respectively) while CuET-treated cells (1μM, 6h) accumulate only the non-cleaved 120-KDa form; b ) Inhibition of the NRF1 cleavage process (appearance of the lower band) by CuET and NMS873 (a p97 inhibitor; 5μM) in mouse NIH3T3 cells co-treated with the proteasome inhibitor MG132 (5μM for 6h); c ) Time-course example images from a FRAP experiment the quantitative analysis of which is shown in Fig. 2g (U-2OS cells, blue boxes mark areas before bleaching, arrows after bleaching); d ) U-2OS cells pre-extracted by TritonX and stained for K-48-polyUb. The Ab signal intensities for cells treated with DMSO, BTZ (1μM), NMS873 (10μM) and CuET (1μM) are analysed by microscopy-based cytometry and plotted below; e ) Western blot analysis of accumulated poly-Ub proteins in ultracentrifugation-separated microsomal fraction from U-2OS cells treated by mock, CuET (1μM), NMS873 (10μM) or BTZ (1μM) for 3h; f ) UPR in U-2OS and MDA-MB-231 cell lines induced by 6-h treatment with CuET (various concentrations) or positive controls (NMS873 5μM, tunicamycin 2μg/ml, thapsigargin 1μM) manifested by increased levels of Xbp1s, ATF4 and p-eIF2a. Panels a-f are representative of two independent experiments.
Figure Legend Snippet: CuET inhibits the p97 pathway and induces cellular unfolded protein response (UPR) a ) Proteasome inhibitor MG132-treated cells (5μM, 6h) accumulate both forms of NRF1 (120- and 110-KDa bands, upper and lower arrows, respectively) while CuET-treated cells (1μM, 6h) accumulate only the non-cleaved 120-KDa form; b ) Inhibition of the NRF1 cleavage process (appearance of the lower band) by CuET and NMS873 (a p97 inhibitor; 5μM) in mouse NIH3T3 cells co-treated with the proteasome inhibitor MG132 (5μM for 6h); c ) Time-course example images from a FRAP experiment the quantitative analysis of which is shown in Fig. 2g (U-2OS cells, blue boxes mark areas before bleaching, arrows after bleaching); d ) U-2OS cells pre-extracted by TritonX and stained for K-48-polyUb. The Ab signal intensities for cells treated with DMSO, BTZ (1μM), NMS873 (10μM) and CuET (1μM) are analysed by microscopy-based cytometry and plotted below; e ) Western blot analysis of accumulated poly-Ub proteins in ultracentrifugation-separated microsomal fraction from U-2OS cells treated by mock, CuET (1μM), NMS873 (10μM) or BTZ (1μM) for 3h; f ) UPR in U-2OS and MDA-MB-231 cell lines induced by 6-h treatment with CuET (various concentrations) or positive controls (NMS873 5μM, tunicamycin 2μg/ml, thapsigargin 1μM) manifested by increased levels of Xbp1s, ATF4 and p-eIF2a. Panels a-f are representative of two independent experiments.

Techniques Used: Inhibition, Staining, Microscopy, Cytometry, Western Blot, Multiple Displacement Amplification

2) Product Images from "A Hypomorphic PALB2 Allele Gives Rise to an Unusual Form of FA-N Associated with Lymphoid Tumour Development"

Article Title: A Hypomorphic PALB2 Allele Gives Rise to an Unusual Form of FA-N Associated with Lymphoid Tumour Development

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005945

Absence of Rad51 foci in fibroblasts from patients II-4 II-5. Following exposure to either 3Gy IR or 50ngml -1 mitomycin C no Rad51 foci were detected in fibroblasts from II-4 and II-5. γH2AX foci indicate the presence of DNA DSB. The residual foci at 24h following IR are consistent with a DNA repair deficiency.
Figure Legend Snippet: Absence of Rad51 foci in fibroblasts from patients II-4 II-5. Following exposure to either 3Gy IR or 50ngml -1 mitomycin C no Rad51 foci were detected in fibroblasts from II-4 and II-5. γH2AX foci indicate the presence of DNA DSB. The residual foci at 24h following IR are consistent with a DNA repair deficiency.

Techniques Used:

3) Product Images from "BRG1 Promotes chromatin remodeling around DNA damage sites"

Article Title: BRG1 Promotes chromatin remodeling around DNA damage sites

Journal: Animal Cells and Systems

doi: 10.1080/19768354.2018.1525429

BRG1 knockdown impairs DNA damage repair. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. siCont: control siRNA. siBRG1: BRG1 siRNA.
Figure Legend Snippet: BRG1 knockdown impairs DNA damage repair. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. siCont: control siRNA. siBRG1: BRG1 siRNA.

Techniques Used: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Software

4) Product Images from "Alcohol-abuse drug disulfiram targets cancer via p97 segregase adapter NPL4"

Article Title: Alcohol-abuse drug disulfiram targets cancer via p97 segregase adapter NPL4

Journal: Nature

doi: 10.1038/nature25016

CuET-induced proteasome inhibition-like response is not due to proteasome inhibition a ) Kinetics of poly-Ub-protein accumulation in U-2OS cells treated with CuET or the proteasome inhibitor BTZ; b ) CuET treatment (1,5h) induces rapid deubiquitylation of ubiquitylated histone H2A (uH2A) similarly to proteasome inhibitors BTZ or MG132 (U-2OS cells); c ) CuET treatment (1,5h) induces rapid cytoplasmic accumulation of poly-ubiquitylated proteins (FK2 antibody staining, U-2OS cells); analogous to BTZ and MG132; d ) 20S proteasome activity is not inhibited by CuET as examined in live MDA-MB-231 cells or e ) in lysates from MDA-MB-231 cells (mean and SD from 4 independent experiments); f ) CuET treatment (1μM, 6h) does not cause accumulation of p53 in the presence of Dicoumarol (300 μM) in MCF7 cells; g ) In-vitro 26S proteasome function measured as Rpn11 deubiquitylation activity, is not inhibited by CuET; 1,10 phenantroline (1,10-OPT) served as a positive control (representative of three independent experiments). Panels a-c, f are representative of two independent experiments.
Figure Legend Snippet: CuET-induced proteasome inhibition-like response is not due to proteasome inhibition a ) Kinetics of poly-Ub-protein accumulation in U-2OS cells treated with CuET or the proteasome inhibitor BTZ; b ) CuET treatment (1,5h) induces rapid deubiquitylation of ubiquitylated histone H2A (uH2A) similarly to proteasome inhibitors BTZ or MG132 (U-2OS cells); c ) CuET treatment (1,5h) induces rapid cytoplasmic accumulation of poly-ubiquitylated proteins (FK2 antibody staining, U-2OS cells); analogous to BTZ and MG132; d ) 20S proteasome activity is not inhibited by CuET as examined in live MDA-MB-231 cells or e ) in lysates from MDA-MB-231 cells (mean and SD from 4 independent experiments); f ) CuET treatment (1μM, 6h) does not cause accumulation of p53 in the presence of Dicoumarol (300 μM) in MCF7 cells; g ) In-vitro 26S proteasome function measured as Rpn11 deubiquitylation activity, is not inhibited by CuET; 1,10 phenantroline (1,10-OPT) served as a positive control (representative of three independent experiments). Panels a-c, f are representative of two independent experiments.

Techniques Used: Inhibition, Staining, Activity Assay, Multiple Displacement Amplification, In Vitro, Positive Control

Related Articles

Western Blot:

Article Title: Topoisomerase II inhibitors induce cGAS-STING dependent inflammation resulting in cytokine induction and immune checkpoint activation
Article Snippet: .. Western blotting Western blotting was carried out as previously described [ ] using the following antibodies/dilutions; anti-γH2AX (1/2000; JBW301-MerckMillipore), anti-β-actin (1/10000; A2228-Sigma), anti-STING (1/1000, 13647-Cell Signalling), anti-cGAS (1/1000; 15102-Cell Signalling), anti-vinculin (1/1000; 14-9777-82-Thermo), anti-mouse-HRP (1/10000; 7076-Cell signalling), anti-rabbit-HRP (1/10000; 7074-Cell Signalling). .. Immunofluorescence Immunofluorescent staining was carried out as previously described [ ] using the following antibodies/dulitions; anti-dsDNA (1/50; Santa Cruz Biotechnology), anti-γH2AX (1/2000; JBW301-MerckMillipore), anti-Lamin B1 (1/1000; ab16048-Abcam), anti-mouse-IgG AlexaFlour-488 (1/1000; ab150113-Abcam), anti-rabbit-IgG AlexaFlour-594 (1/1000; A-11072-ThemoFischer), anti-mouse-IgG AlexaFlour-594 (1:1000; ReadyProbes).

Incubation:

Article Title: Analyzing the influence of kinase inhibitors on DNA repair by differential proteomics of chromatin-interacting proteins and nuclear phospho-proteins
Article Snippet: .. Another 24 h later cells replated and were fixed, permeabilized, blocked and incubated with anti-γH2AX (Merck Millipore, 05–636) and anti-53BP1 (Novus Biologicals) primary and secondary antibodies (Fluorescein-labeled anti-mouse: red, life technologies, A11005; anti-rabbit: green, GE-Healthcare, Amersham™) 24 h later. .. Cell nuclei were stained with DAPI (QBiogene).

Article Title: In Vivo Stabilization of a Gastrin-Releasing Peptide Receptor Antagonist Enhances PET Imaging and Radionuclide Therapy of Prostate Cancer in Preclinical Studies
Article Snippet: .. Primary antibodies [anti-GEMININ (10802-1-AP, Proteintech Group, 1/400 dilution), anti-53BP1 (NB100-304, Novus Biologicals, dilution 1/1000), or anti-γH2AX (05-636, Merck-Millipore, dilution 1/500)] were diluted in blocking buffer and incubated for 90 min at room temperature (RT). .. Secondary antibodies (Alexa Fluor 594 or 488, Life Technologies; dilution 1/1000) were diluted in blocking buffer and incubated for 60 min at RT.

Blocking Assay:

Article Title: In Vivo Stabilization of a Gastrin-Releasing Peptide Receptor Antagonist Enhances PET Imaging and Radionuclide Therapy of Prostate Cancer in Preclinical Studies
Article Snippet: .. Primary antibodies [anti-GEMININ (10802-1-AP, Proteintech Group, 1/400 dilution), anti-53BP1 (NB100-304, Novus Biologicals, dilution 1/1000), or anti-γH2AX (05-636, Merck-Millipore, dilution 1/500)] were diluted in blocking buffer and incubated for 90 min at room temperature (RT). .. Secondary antibodies (Alexa Fluor 594 or 488, Life Technologies; dilution 1/1000) were diluted in blocking buffer and incubated for 60 min at RT.

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