Structured Review

Merck KGaA gapdh
The <t>p53</t> synthesis efficiency under different stress conditions in NIH3T3 cells. The cells were exposed for 16 hours to doxorubicin (0.1 μM), tunicamycin (1.2 μM) and thapsigargin (0.1 μM), or to an equivalent volume of DMSO, and then harvested. Endogenous p53 protein level was determined by western blot (a) or flow cytometry (b) using monoclonal 1C12 antibody. <t>GAPDH</t> was used as a loading control. The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) was also conducted. For RT-PCR analysis, the samples were prepared using total RNA extracted from mouse fibroblasts treated with DMSO as a control, tunicamycin, thapsigargin and doxorubicin.
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Images

1) Product Images from "Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies"

Article Title: Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

Journal: RNA Biology

doi: 10.1080/15476286.2018.1556084

The p53 synthesis efficiency under different stress conditions in NIH3T3 cells. The cells were exposed for 16 hours to doxorubicin (0.1 μM), tunicamycin (1.2 μM) and thapsigargin (0.1 μM), or to an equivalent volume of DMSO, and then harvested. Endogenous p53 protein level was determined by western blot (a) or flow cytometry (b) using monoclonal 1C12 antibody. GAPDH was used as a loading control. The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) was also conducted. For RT-PCR analysis, the samples were prepared using total RNA extracted from mouse fibroblasts treated with DMSO as a control, tunicamycin, thapsigargin and doxorubicin.
Figure Legend Snippet: The p53 synthesis efficiency under different stress conditions in NIH3T3 cells. The cells were exposed for 16 hours to doxorubicin (0.1 μM), tunicamycin (1.2 μM) and thapsigargin (0.1 μM), or to an equivalent volume of DMSO, and then harvested. Endogenous p53 protein level was determined by western blot (a) or flow cytometry (b) using monoclonal 1C12 antibody. GAPDH was used as a loading control. The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) was also conducted. For RT-PCR analysis, the samples were prepared using total RNA extracted from mouse fibroblasts treated with DMSO as a control, tunicamycin, thapsigargin and doxorubicin.

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

2) Product Images from "Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation"

Article Title: Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

Journal: Journal of Virology

doi: 10.1128/JVI.00960-16

Assessment of the interaction of hCKα with NS5A. (A) Huh7 cells coexpressing T7 RNA polymerase and NS3-NS5B were lysed and subjected to co-IP with an anti-NS5 MAb or an isotype-matched control mouse IgG. The precipitates were analyzed by SDS-PAGE, followed by immunoblotting analysis to detect each of the indicated proteins. A portion of aliquots containing 5% of the total proteins in the lysates used for precipitation was loaded as the input control. (B) 293T cells were cotransfected with plasmids encoding each of HCV genotype 1b Con1 strain NS proteins, as indicated, with a pCMV6 plasmid encoding hCKα-Flag. Cell lysates were pulled down with rabbit anti-HA, and the precipitated proteins were subjected to immunoblot analysis with the indicated MAb antibodies. (C) Lysates from Huh7 cells expressing the indicated proteins were precipitated with rabbit anti-Flag, and the coprecipitated proteins were subjected to immunoblot detection with the indicated MAbs. (D) Lysates from Huh7 cells coexpressing GFP-tagged hCKα and different Myc-tagged NS5A domains were pulled down with rabbit anti-GFP, followed by immunoblot analysis using the indicated MAbs.
Figure Legend Snippet: Assessment of the interaction of hCKα with NS5A. (A) Huh7 cells coexpressing T7 RNA polymerase and NS3-NS5B were lysed and subjected to co-IP with an anti-NS5 MAb or an isotype-matched control mouse IgG. The precipitates were analyzed by SDS-PAGE, followed by immunoblotting analysis to detect each of the indicated proteins. A portion of aliquots containing 5% of the total proteins in the lysates used for precipitation was loaded as the input control. (B) 293T cells were cotransfected with plasmids encoding each of HCV genotype 1b Con1 strain NS proteins, as indicated, with a pCMV6 plasmid encoding hCKα-Flag. Cell lysates were pulled down with rabbit anti-HA, and the precipitated proteins were subjected to immunoblot analysis with the indicated MAb antibodies. (C) Lysates from Huh7 cells expressing the indicated proteins were precipitated with rabbit anti-Flag, and the coprecipitated proteins were subjected to immunoblot detection with the indicated MAbs. (D) Lysates from Huh7 cells coexpressing GFP-tagged hCKα and different Myc-tagged NS5A domains were pulled down with rabbit anti-GFP, followed by immunoblot analysis using the indicated MAbs.

Techniques Used: Co-Immunoprecipitation Assay, SDS Page, Plasmid Preparation, Expressing

Analysis of the effect of hCKα activity on ER localization of hCKα and NS5A. (A) The hCKα stable knockdown Huh7 cells were cotransfected with pWPI-T7-BLR along with wild-type or D288A mutant hCKα-R plasmid in the presence or absence of pTM-NS3-NS5B as indicated. The cells were then analyzed by confocal microscopy for the intracellular localization of hCKα and NS5A on the ER. The white boxed areas in merged images were enlarged (Zoom) to show the localization of hCKα and NS5A on the ER (white arrows and white arrowheads, respectively) and the colocalization of hCKα and NS5A on the ER (yellow arrows). (B) The degree of localization of hCKα, NS5A, and hCKα-associated NS5A on the ER was quantified. (C) A representative set of Western blot results for the indicated proteins is shown. ****, P
Figure Legend Snippet: Analysis of the effect of hCKα activity on ER localization of hCKα and NS5A. (A) The hCKα stable knockdown Huh7 cells were cotransfected with pWPI-T7-BLR along with wild-type or D288A mutant hCKα-R plasmid in the presence or absence of pTM-NS3-NS5B as indicated. The cells were then analyzed by confocal microscopy for the intracellular localization of hCKα and NS5A on the ER. The white boxed areas in merged images were enlarged (Zoom) to show the localization of hCKα and NS5A on the ER (white arrows and white arrowheads, respectively) and the colocalization of hCKα and NS5A on the ER (yellow arrows). (B) The degree of localization of hCKα, NS5A, and hCKα-associated NS5A on the ER was quantified. (C) A representative set of Western blot results for the indicated proteins is shown. ****, P

Techniques Used: Activity Assay, Mutagenesis, Plasmid Preparation, Confocal Microscopy, Western Blot

Requirement of hCKα for the binding of NS5A to NS5B in the viral RC. (A) Huh7 cells were cotransfected with pTM-NS3-5B and pWPI-T7-BLR along with pCMV-Flag-NS5B. The transfected cells were fixed and processed by confocal microscopy using NS5A MAb and rabbit anti-Flag (left panel). The boxed area in the NS5A/Flag-NS5B panel was enlarged (Zoom) to show the colocalization of NS5A with Flag-NS5B (white arrowheads). The degree of colocalization of NS5A with Flag-NS5B was quantified (right panel). (B) Cell lysates from the paired control and hCKα stable knockdown Huh7 cells cotransfected with pTM-NS3-NS5B and pWPI-T7-BLR were subjected to co-IP analysis with anti-NS5A MAb, and the precipitated proteins were analyzed by Western blotting for the indicated proteins. (C to E) The paired stable knockdown cells were cotransfected with pCMV plasmids encoding NS5A-Myc and Flag-tagged NS5B (C), NS3 (D), or NS4B (E). The cell lysates were subjected to co-IP with anti-Myc to determine the binding of NS5A to each of the NS proteins. (F) The paired knockdown Huh7 cells were transfected with plasmids encoding pCMV-Flag-NS4B, pWPI-T7-BLR, and pTM-NS5B. Cell lysates were precipitated with anti-Flag to determine the binding of NS4B to NS5B. ****, P
Figure Legend Snippet: Requirement of hCKα for the binding of NS5A to NS5B in the viral RC. (A) Huh7 cells were cotransfected with pTM-NS3-5B and pWPI-T7-BLR along with pCMV-Flag-NS5B. The transfected cells were fixed and processed by confocal microscopy using NS5A MAb and rabbit anti-Flag (left panel). The boxed area in the NS5A/Flag-NS5B panel was enlarged (Zoom) to show the colocalization of NS5A with Flag-NS5B (white arrowheads). The degree of colocalization of NS5A with Flag-NS5B was quantified (right panel). (B) Cell lysates from the paired control and hCKα stable knockdown Huh7 cells cotransfected with pTM-NS3-NS5B and pWPI-T7-BLR were subjected to co-IP analysis with anti-NS5A MAb, and the precipitated proteins were analyzed by Western blotting for the indicated proteins. (C to E) The paired stable knockdown cells were cotransfected with pCMV plasmids encoding NS5A-Myc and Flag-tagged NS5B (C), NS3 (D), or NS4B (E). The cell lysates were subjected to co-IP with anti-Myc to determine the binding of NS5A to each of the NS proteins. (F) The paired knockdown Huh7 cells were transfected with plasmids encoding pCMV-Flag-NS4B, pWPI-T7-BLR, and pTM-NS5B. Cell lysates were precipitated with anti-Flag to determine the binding of NS4B to NS5B. ****, P

Techniques Used: Binding Assay, Transfection, Confocal Microscopy, Co-Immunoprecipitation Assay, Western Blot

Involvement of hCKα in HCV replication. (A) Biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). PC is mainly synthesized via the CDP-choline and PE methyltransferase (PEMT) pathways. In the CDP-choline pathway, choline kinase (CK), CTP:phosphocholine cytidylyltransferase (CCT), and choline phosphotransferase (CPT) catalyze consecutive reactions to produce PC. In the PEMT pathway, PE undergoes three methylation reactions catalyzed by PEMT to synthesize PC. In the CDP-ethanolamine pathway, ethanolamine is taken up into the cytoplasm and catalyzed by ethanolamine kinase (EK), CTP:phosphoethanolamine cytidylyltransferase (ECT), and ethanolamine phosphotransferase (EPT) to yield PE. (B and C) Huh7 cells remained untransfected (−) or were transfected with an untargeted control siRNA or hCKα-specific siRNA. Cells were then infected with HCVcc at an MOI of 1 or received parallel treatment without HCVcc (−). Cells were then subjected to Western blot analysis (B) and RT-PCR (C). (D) Huh7 cells transfected with control or hCKα siRNAs (left panel) and Huh7 cells stably transduced with control or hCKα shRNA lentiviral vectors (right panel) were assessed for cell viability. (E) The control and hCKα shRNA Huh7 stable cells were transfected with plasmids encoding the Flag-tagged normal hCKα (α) or shRNA-resistant hCKα (α-R) followed by infection with HCV. The cells were analyzed by Western blotting (top panel). The levels of NS3, NS5A, and core proteins in HCV-infected control or hCKα stable knockdown cells overexpressing hCKα or hCKα-R are expressed as a percentage relative to that detected in vector plasmid transfected cells, which was designated 100% (bottom panel). *, P
Figure Legend Snippet: Involvement of hCKα in HCV replication. (A) Biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). PC is mainly synthesized via the CDP-choline and PE methyltransferase (PEMT) pathways. In the CDP-choline pathway, choline kinase (CK), CTP:phosphocholine cytidylyltransferase (CCT), and choline phosphotransferase (CPT) catalyze consecutive reactions to produce PC. In the PEMT pathway, PE undergoes three methylation reactions catalyzed by PEMT to synthesize PC. In the CDP-ethanolamine pathway, ethanolamine is taken up into the cytoplasm and catalyzed by ethanolamine kinase (EK), CTP:phosphoethanolamine cytidylyltransferase (ECT), and ethanolamine phosphotransferase (EPT) to yield PE. (B and C) Huh7 cells remained untransfected (−) or were transfected with an untargeted control siRNA or hCKα-specific siRNA. Cells were then infected with HCVcc at an MOI of 1 or received parallel treatment without HCVcc (−). Cells were then subjected to Western blot analysis (B) and RT-PCR (C). (D) Huh7 cells transfected with control or hCKα siRNAs (left panel) and Huh7 cells stably transduced with control or hCKα shRNA lentiviral vectors (right panel) were assessed for cell viability. (E) The control and hCKα shRNA Huh7 stable cells were transfected with plasmids encoding the Flag-tagged normal hCKα (α) or shRNA-resistant hCKα (α-R) followed by infection with HCV. The cells were analyzed by Western blotting (top panel). The levels of NS3, NS5A, and core proteins in HCV-infected control or hCKα stable knockdown cells overexpressing hCKα or hCKα-R are expressed as a percentage relative to that detected in vector plasmid transfected cells, which was designated 100% (bottom panel). *, P

Techniques Used: Synthesized, Cycling Probe Technology, Methylation, Transfection, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transduction, shRNA, Plasmid Preparation

Abrogation of HCV-induced MW formation by hCKα depletion or CK37 treatment. (A) Control shRNA and hCKα shRNA stable expression cells were transfected with pTM-NS3-5B and pWPI-T7-BLR, fixed, and processed for TEM. (B) Huh7 cells were first cotransfected with plasmids expressing T7 polymerase and NS3-NS5B. At 24 h post-DNA transfection, cells were left untreated or treated with 100 μM CK37 for 18 h, and then cells were analyzed by TEM for MW formation. The area boxed in red was enlarged as shown. Light blue arrows indicate vesicles of heterogeneous sizes (left panels of A and B) and small vesicles in CK37-treated Huh7 cells (right panel of B); blue arrowheads indicate double-membrane vesicles (DMVs) (left panels of A and B). N, nucleus; LD, lipid droplet; ER, endoplasmic reticulum; M, mitochondria. (C) Thirty cells randomly picked from each experimental setting were examined by TEM, and the numbers of cells showing three different phenotypes were quantified. MW, vesicles 130 to 270 nm in diameter; small vesicles, vesicles 40 to 100 nm in diameter.
Figure Legend Snippet: Abrogation of HCV-induced MW formation by hCKα depletion or CK37 treatment. (A) Control shRNA and hCKα shRNA stable expression cells were transfected with pTM-NS3-5B and pWPI-T7-BLR, fixed, and processed for TEM. (B) Huh7 cells were first cotransfected with plasmids expressing T7 polymerase and NS3-NS5B. At 24 h post-DNA transfection, cells were left untreated or treated with 100 μM CK37 for 18 h, and then cells were analyzed by TEM for MW formation. The area boxed in red was enlarged as shown. Light blue arrows indicate vesicles of heterogeneous sizes (left panels of A and B) and small vesicles in CK37-treated Huh7 cells (right panel of B); blue arrowheads indicate double-membrane vesicles (DMVs) (left panels of A and B). N, nucleus; LD, lipid droplet; ER, endoplasmic reticulum; M, mitochondria. (C) Thirty cells randomly picked from each experimental setting were examined by TEM, and the numbers of cells showing three different phenotypes were quantified. MW, vesicles 130 to 270 nm in diameter; small vesicles, vesicles 40 to 100 nm in diameter.

Techniques Used: shRNA, Expressing, Transfection, Transmission Electron Microscopy

A model illustrating the function of hCKα in HCV RNA replication. HCV usurps hCKα to promote viral RNA replication by facilitating functional viral RC formation on the ER. hCKα is first recruited by HCV, likely via its interaction with NS5A D1; hCKα activity then mediates the targeting of the hCKα-NS5A complex to the ER, whereas expression of NS3-NS5B also enhances the accumulation of the hCKα-NS5A complex on the ER. On the ER membrane, the hCKα protein, per se , triggers NS5A and NS5B interactions, resulting in functional viral RC assembly and MW formation. These events subsequently promote viral RNA replication, which subsequently leads to extended MW formation. The steps that involve hCKα activity or protein (not activity) are indicated by red arrows.
Figure Legend Snippet: A model illustrating the function of hCKα in HCV RNA replication. HCV usurps hCKα to promote viral RNA replication by facilitating functional viral RC formation on the ER. hCKα is first recruited by HCV, likely via its interaction with NS5A D1; hCKα activity then mediates the targeting of the hCKα-NS5A complex to the ER, whereas expression of NS3-NS5B also enhances the accumulation of the hCKα-NS5A complex on the ER. On the ER membrane, the hCKα protein, per se , triggers NS5A and NS5B interactions, resulting in functional viral RC assembly and MW formation. These events subsequently promote viral RNA replication, which subsequently leads to extended MW formation. The steps that involve hCKα activity or protein (not activity) are indicated by red arrows.

Techniques Used: Functional Assay, Activity Assay, Expressing

The effect of hCKα knockdown on ER localization of NS5A, NS5B, and NS3. The control and hCKα shRNA Huh7 stable cells were transfected with pTM-NS3-5B and pWPI-T7-BLR, and the cells were examined by confocal microscopy for ER localization of NS5A (A), NS5B (B), and NS3 (C) (left panels). The white boxed areas in the NS/CALR images were enlarged (Zoom), and white arrowheads indicate the ER localization of each indicated NS protein. The extent of colocalization of each NS protein with CALR in control and hCKα shRNA cells was quantified (right panel). ****, P
Figure Legend Snippet: The effect of hCKα knockdown on ER localization of NS5A, NS5B, and NS3. The control and hCKα shRNA Huh7 stable cells were transfected with pTM-NS3-5B and pWPI-T7-BLR, and the cells were examined by confocal microscopy for ER localization of NS5A (A), NS5B (B), and NS3 (C) (left panels). The white boxed areas in the NS/CALR images were enlarged (Zoom), and white arrowheads indicate the ER localization of each indicated NS protein. The extent of colocalization of each NS protein with CALR in control and hCKα shRNA cells was quantified (right panel). ****, P

Techniques Used: shRNA, Transfection, Confocal Microscopy

3) Product Images from "Distribution of oxidized DJ-1 in Parkinson’s disease-related sites in the brain and in the peripheral tissues: effects of aging and a neurotoxin"

Article Title: Distribution of oxidized DJ-1 in Parkinson’s disease-related sites in the brain and in the peripheral tissues: effects of aging and a neurotoxin

Journal: Scientific Reports

doi: 10.1038/s41598-018-30561-z

The effects of aging on oxDJ-1 levels in the brain. (a) Western blot analyses of oxDJ-1 in separated brain tissues of aged mice. Protein lysates of each brain site were subjected to western blot analyses. The relative band densities of oxDJ-1 normalized to DJ-1 were calculated and are presented as mean (n = 2). Abbreviations are same with Fig. 1 . ( b – d ) Protein lysates of the substantia nigra (SN, b ), striatum (Str, c ), and olfactory bulb (OB, d ) of young (9 weeks) and aged (130 weeks) WT mice were separated by SDS-PAGE and then subjected to western blotting using each Ab. The densities of each band were determined and the relative densities of oxDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (n = 3). * P
Figure Legend Snippet: The effects of aging on oxDJ-1 levels in the brain. (a) Western blot analyses of oxDJ-1 in separated brain tissues of aged mice. Protein lysates of each brain site were subjected to western blot analyses. The relative band densities of oxDJ-1 normalized to DJ-1 were calculated and are presented as mean (n = 2). Abbreviations are same with Fig. 1 . ( b – d ) Protein lysates of the substantia nigra (SN, b ), striatum (Str, c ), and olfactory bulb (OB, d ) of young (9 weeks) and aged (130 weeks) WT mice were separated by SDS-PAGE and then subjected to western blotting using each Ab. The densities of each band were determined and the relative densities of oxDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (n = 3). * P

Techniques Used: Western Blot, Mouse Assay, SDS Page

Elevation of oxidized DJ-1 in the brain and heart of MPTP-treated mice. After the administration of PBS and MPTP (15 mg/kg, 3 times i.p.), each brain and heart were separated and subjected to analysis. (a–d) Protein lysates of the substantia nigra (SN, a ), striatum (Str, b ), olfactory bulb (OB, c ), and heart ( d ) of MPTP-treated mice were subjected to western blot analyses using each specific Ab. The relative band densities of oxDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (n = 6). ** P
Figure Legend Snippet: Elevation of oxidized DJ-1 in the brain and heart of MPTP-treated mice. After the administration of PBS and MPTP (15 mg/kg, 3 times i.p.), each brain and heart were separated and subjected to analysis. (a–d) Protein lysates of the substantia nigra (SN, a ), striatum (Str, b ), olfactory bulb (OB, c ), and heart ( d ) of MPTP-treated mice were subjected to western blot analyses using each specific Ab. The relative band densities of oxDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (n = 6). ** P

Techniques Used: Mouse Assay, Western Blot

Change of oxDJ-1 levels in the heart and skeletal muscle of aged mice. (a) Lysates from each tissue of young (9 weeks) and aged (130 weeks) wild-type (WT) mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The change in oxDJ-1 levels in the skeletal muscle and heart is shown. (b–e) Proteins in lysates from hearts (b and c) and skeletal muscle (d and e) of young and aged WT mice were separated by SDS-PAGE (b and d) or 2D-PAGE (c and e) and then subjected to western blotting using the indicated Ab. The densities of each band were determined and the relative densities of oxDJ-1 compared with DJ-1 were calculated and are presented as mean ± SD (b, n = 3). In 2D-PAGE, black, white, and striped arrowheads indicate native DJ-1, oxDJ-1, and unknown modified DJ-1 (UmDJ-1). The densities of each spot were determined and the relative densities of oxDJ-1 and UmDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (C, n = 3). * P
Figure Legend Snippet: Change of oxDJ-1 levels in the heart and skeletal muscle of aged mice. (a) Lysates from each tissue of young (9 weeks) and aged (130 weeks) wild-type (WT) mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The change in oxDJ-1 levels in the skeletal muscle and heart is shown. (b–e) Proteins in lysates from hearts (b and c) and skeletal muscle (d and e) of young and aged WT mice were separated by SDS-PAGE (b and d) or 2D-PAGE (c and e) and then subjected to western blotting using the indicated Ab. The densities of each band were determined and the relative densities of oxDJ-1 compared with DJ-1 were calculated and are presented as mean ± SD (b, n = 3). In 2D-PAGE, black, white, and striped arrowheads indicate native DJ-1, oxDJ-1, and unknown modified DJ-1 (UmDJ-1). The densities of each spot were determined and the relative densities of oxDJ-1 and UmDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (C, n = 3). * P

Techniques Used: Mouse Assay, Western Blot, SDS Page, Polyacrylamide Gel Electrophoresis, Modification

Distribution of oxidized DJ-1 in mouse brain and peripheral tissues. (a) Lysates from each tissue of young wild-type (WT) and DJ-1 −/− mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The specific oxDJ-1 band is indicated by a black arrowhead and a nonspecific band is indicated by an asterisk. (b) Comparison of oxDJ-1 immunoreactivity with TH immunostaining in the olfactory bulb (OB), substantia nigra (SN), and striatum (Str). Sagittal sections from wild-type C57BL/6J mice were stained with anti-oxDJ-1 Ab and anti-TH Ab. Oxidized DJ-1 IR was observed around TH-positive neurons. (c,d) Confocal images of oxDJ-1 in SN dopaminergic neurons and astrocytes. A section containing the SN was immunostained with anti-oxDJ-1 Ab and anti-TH Ab (c) or anti-glial fibrillary acidic protein (GFAP) Ab (d) and then visualized using fluorescence confocal microscopy. A scale bar is shown in each figure. (e) Western blot analyses of oxDJ-1 in separated brain tissues. Protein lysates of each brain site were subjected to western blot analyses using Abs against DJ-1, oxDJ-1, TH, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative band densities of oxDJ-1 normalized to DJ-1 were calculated and are presented as mean ± SD (n = 6). * P
Figure Legend Snippet: Distribution of oxidized DJ-1 in mouse brain and peripheral tissues. (a) Lysates from each tissue of young wild-type (WT) and DJ-1 −/− mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The specific oxDJ-1 band is indicated by a black arrowhead and a nonspecific band is indicated by an asterisk. (b) Comparison of oxDJ-1 immunoreactivity with TH immunostaining in the olfactory bulb (OB), substantia nigra (SN), and striatum (Str). Sagittal sections from wild-type C57BL/6J mice were stained with anti-oxDJ-1 Ab and anti-TH Ab. Oxidized DJ-1 IR was observed around TH-positive neurons. (c,d) Confocal images of oxDJ-1 in SN dopaminergic neurons and astrocytes. A section containing the SN was immunostained with anti-oxDJ-1 Ab and anti-TH Ab (c) or anti-glial fibrillary acidic protein (GFAP) Ab (d) and then visualized using fluorescence confocal microscopy. A scale bar is shown in each figure. (e) Western blot analyses of oxDJ-1 in separated brain tissues. Protein lysates of each brain site were subjected to western blot analyses using Abs against DJ-1, oxDJ-1, TH, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative band densities of oxDJ-1 normalized to DJ-1 were calculated and are presented as mean ± SD (n = 6). * P

Techniques Used: Mouse Assay, Western Blot, Immunostaining, Staining, Fluorescence, Confocal Microscopy

4) Product Images from "Canonical and Kinase Activity-Independent Mechanisms for Extracellular Signal-Regulated Kinase 5 (ERK5) Nuclear Translocation Require Dissociation of Hsp90 from the ERK5-Cdc37 Complex"

Article Title: Canonical and Kinase Activity-Independent Mechanisms for Extracellular Signal-Regulated Kinase 5 (ERK5) Nuclear Translocation Require Dissociation of Hsp90 from the ERK5-Cdc37 Complex

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01246-12

ERK5 associates with Hsp90 and cochaperone Cdc37 through its N-terminal half. (A) One milligram of SH-SY5Y lysates was immunoprecipitated (IP) with anti-Cdc37, anti-ERK5, or anti-Hsp90β antibodies followed by immunoblotting of the immune complexes.
Figure Legend Snippet: ERK5 associates with Hsp90 and cochaperone Cdc37 through its N-terminal half. (A) One milligram of SH-SY5Y lysates was immunoprecipitated (IP) with anti-Cdc37, anti-ERK5, or anti-Hsp90β antibodies followed by immunoblotting of the immune complexes.

Techniques Used: Immunoprecipitation

ERK5 activation induces Hsp90β dissociation from the ERK5-Cdc37 complex. (A) HeLa cells were starved for 16 h prior to stimulation with EGF. One milligram of lysate was immunoprecipitated with anti-ERK5 (left panels) or anti-Akt (right panels)
Figure Legend Snippet: ERK5 activation induces Hsp90β dissociation from the ERK5-Cdc37 complex. (A) HeLa cells were starved for 16 h prior to stimulation with EGF. One milligram of lysate was immunoprecipitated with anti-ERK5 (left panels) or anti-Akt (right panels)

Techniques Used: Activation Assay, Immunoprecipitation

Cdc37 overexpression induces Hsp90 dissociation from the ERK5-Cdc37 complex and nuclear translocation of ERK5. (A) Cells were transfected with 1 μg of plasmids encoding GST-ERK5 and/or Hsp90β and the indicated amounts of plasmid encoding
Figure Legend Snippet: Cdc37 overexpression induces Hsp90 dissociation from the ERK5-Cdc37 complex and nuclear translocation of ERK5. (A) Cells were transfected with 1 μg of plasmids encoding GST-ERK5 and/or Hsp90β and the indicated amounts of plasmid encoding

Techniques Used: Over Expression, Translocation Assay, Transfection, Plasmid Preparation

5) Product Images from "ANTIOXIDANTS PROTECT CALSEQUESTRIN-1 KNOCKOUT MICE FROM HALOTHANE- AND HEAT-INDUCED SUDDEN DEATH"

Article Title: ANTIOXIDANTS PROTECT CALSEQUESTRIN-1 KNOCKOUT MICE FROM HALOTHANE- AND HEAT-INDUCED SUDDEN DEATH

Journal: Anesthesiology

doi: 10.1097/ALN.0000000000000748

NAC treatment reduces SOD1 expression, 3-nitrotyrosylation (3-NT), and the increased oxidative stress of EDL muscles from CASQ1-null mice A) Representative immunoblots showing expression levels of SOD1 in EDL muscles from untreated WT (n = 7), NAC-treated WT (n = 5), untreated CASQ1-null (n = 8), and NAC-treated CASQ1-null (n = 8) mice. B) Relative band densities, expressed as SOD1/α-actinin ratio, show that SOD-1 expression is significantly increased in EDL muscles of CASQ1-null mice compared to that for both untreated WT and NAC-treated CASQ1-null mice. SOD1 expression was not significantly different between EDL muscles from untreated and NAC-treated WT mice. C) Representative immunoblots showing levels of 3-nitrotyrosylation (3-NT) in untreated WT (n = 6), NAC-treated WT (n = 5), untreated CASQ1-null (n = 6), and NAC-treated CASQ-null (n = 6) mice. D) Relative band densities, expressed as 3-NT/GAPDH ratio, show that levels of 3-NT are significantly increased in EDL muscles from CASQ1-null mice compared to that of WT mice. NAC treatment significantly reduced levels of 3-NT in EDL muscles from both WT and CASQ1-null mice. E-F) Values of reduced glutathione (GSH, expressed as nmol/g of tissue) and GSH/GSSG ratio are significantly reduced in EDL muscle homogenates from CASQ1-null mice compared to that from WT and are significantly increased after treatment with NAC. Data are given as means ± SEM; n = number of mice. Abbreviations: 3-NT, 3-nitrotyrosine; CASQ1-null, Calsequestrin-1 knockout; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; NAC, N-acetylcysteine; SOD1, superoxide dismutase type-1; WT, wild type.
Figure Legend Snippet: NAC treatment reduces SOD1 expression, 3-nitrotyrosylation (3-NT), and the increased oxidative stress of EDL muscles from CASQ1-null mice A) Representative immunoblots showing expression levels of SOD1 in EDL muscles from untreated WT (n = 7), NAC-treated WT (n = 5), untreated CASQ1-null (n = 8), and NAC-treated CASQ1-null (n = 8) mice. B) Relative band densities, expressed as SOD1/α-actinin ratio, show that SOD-1 expression is significantly increased in EDL muscles of CASQ1-null mice compared to that for both untreated WT and NAC-treated CASQ1-null mice. SOD1 expression was not significantly different between EDL muscles from untreated and NAC-treated WT mice. C) Representative immunoblots showing levels of 3-nitrotyrosylation (3-NT) in untreated WT (n = 6), NAC-treated WT (n = 5), untreated CASQ1-null (n = 6), and NAC-treated CASQ-null (n = 6) mice. D) Relative band densities, expressed as 3-NT/GAPDH ratio, show that levels of 3-NT are significantly increased in EDL muscles from CASQ1-null mice compared to that of WT mice. NAC treatment significantly reduced levels of 3-NT in EDL muscles from both WT and CASQ1-null mice. E-F) Values of reduced glutathione (GSH, expressed as nmol/g of tissue) and GSH/GSSG ratio are significantly reduced in EDL muscle homogenates from CASQ1-null mice compared to that from WT and are significantly increased after treatment with NAC. Data are given as means ± SEM; n = number of mice. Abbreviations: 3-NT, 3-nitrotyrosine; CASQ1-null, Calsequestrin-1 knockout; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; NAC, N-acetylcysteine; SOD1, superoxide dismutase type-1; WT, wild type.

Techniques Used: Expressing, Mouse Assay, Western Blot, Knock-Out

6) Product Images from "Distribution of oxidized DJ-1 in Parkinson’s disease-related sites in the brain and in the peripheral tissues: effects of aging and a neurotoxin"

Article Title: Distribution of oxidized DJ-1 in Parkinson’s disease-related sites in the brain and in the peripheral tissues: effects of aging and a neurotoxin

Journal: Scientific Reports

doi: 10.1038/s41598-018-30561-z

Elevation of oxidized DJ-1 in the brain and heart of MPTP-treated mice. After the administration of PBS and MPTP (15 mg/kg, 3 times i.p.), each brain and heart were separated and subjected to analysis. (a–d) Protein lysates of the substantia nigra (SN, a ), striatum (Str, b ), olfactory bulb (OB, c ), and heart ( d ) of MPTP-treated mice were subjected to western blot analyses using each specific Ab. The relative band densities of oxDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (n = 6). ** P
Figure Legend Snippet: Elevation of oxidized DJ-1 in the brain and heart of MPTP-treated mice. After the administration of PBS and MPTP (15 mg/kg, 3 times i.p.), each brain and heart were separated and subjected to analysis. (a–d) Protein lysates of the substantia nigra (SN, a ), striatum (Str, b ), olfactory bulb (OB, c ), and heart ( d ) of MPTP-treated mice were subjected to western blot analyses using each specific Ab. The relative band densities of oxDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (n = 6). ** P

Techniques Used: Mouse Assay, Western Blot

Change of oxDJ-1 levels in the heart and skeletal muscle of aged mice. (a) Lysates from each tissue of young (9 weeks) and aged (130 weeks) wild-type (WT) mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The change in oxDJ-1 levels in the skeletal muscle and heart is shown. (b–e) Proteins in lysates from hearts (b and c) and skeletal muscle (d and e) of young and aged WT mice were separated by SDS-PAGE (b and d) or 2D-PAGE (c and e) and then subjected to western blotting using the indicated Ab. The densities of each band were determined and the relative densities of oxDJ-1 compared with DJ-1 were calculated and are presented as mean ± SD (b, n = 3). In 2D-PAGE, black, white, and striped arrowheads indicate native DJ-1, oxDJ-1, and unknown modified DJ-1 (UmDJ-1). The densities of each spot were determined and the relative densities of oxDJ-1 and UmDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (C, n = 3). * P
Figure Legend Snippet: Change of oxDJ-1 levels in the heart and skeletal muscle of aged mice. (a) Lysates from each tissue of young (9 weeks) and aged (130 weeks) wild-type (WT) mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The change in oxDJ-1 levels in the skeletal muscle and heart is shown. (b–e) Proteins in lysates from hearts (b and c) and skeletal muscle (d and e) of young and aged WT mice were separated by SDS-PAGE (b and d) or 2D-PAGE (c and e) and then subjected to western blotting using the indicated Ab. The densities of each band were determined and the relative densities of oxDJ-1 compared with DJ-1 were calculated and are presented as mean ± SD (b, n = 3). In 2D-PAGE, black, white, and striped arrowheads indicate native DJ-1, oxDJ-1, and unknown modified DJ-1 (UmDJ-1). The densities of each spot were determined and the relative densities of oxDJ-1 and UmDJ-1 relative to DJ-1 were calculated and are presented as mean ± SD (C, n = 3). * P

Techniques Used: Mouse Assay, Western Blot, SDS Page, Polyacrylamide Gel Electrophoresis, Modification

Distribution of oxidized DJ-1 in mouse brain and peripheral tissues. (a) Lysates from each tissue of young wild-type (WT) and DJ-1 −/− mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The specific oxDJ-1 band is indicated by a black arrowhead and a nonspecific band is indicated by an asterisk. (b) Comparison of oxDJ-1 immunoreactivity with TH immunostaining in the olfactory bulb (OB), substantia nigra (SN), and striatum (Str). Sagittal sections from wild-type C57BL/6J mice were stained with anti-oxDJ-1 Ab and anti-TH Ab. Oxidized DJ-1 IR was observed around TH-positive neurons. (c,d) Confocal images of oxDJ-1 in SN dopaminergic neurons and astrocytes. A section containing the SN was immunostained with anti-oxDJ-1 Ab and anti-TH Ab (c) or anti-glial fibrillary acidic protein (GFAP) Ab (d) and then visualized using fluorescence confocal microscopy. A scale bar is shown in each figure. (e) Western blot analyses of oxDJ-1 in separated brain tissues. Protein lysates of each brain site were subjected to western blot analyses using Abs against DJ-1, oxDJ-1, TH, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative band densities of oxDJ-1 normalized to DJ-1 were calculated and are presented as mean ± SD (n = 6). * P
Figure Legend Snippet: Distribution of oxidized DJ-1 in mouse brain and peripheral tissues. (a) Lysates from each tissue of young wild-type (WT) and DJ-1 −/− mice were subjected to western blotting using anti-oxDJ-1 and anti-DJ-1 Abs. The specific oxDJ-1 band is indicated by a black arrowhead and a nonspecific band is indicated by an asterisk. (b) Comparison of oxDJ-1 immunoreactivity with TH immunostaining in the olfactory bulb (OB), substantia nigra (SN), and striatum (Str). Sagittal sections from wild-type C57BL/6J mice were stained with anti-oxDJ-1 Ab and anti-TH Ab. Oxidized DJ-1 IR was observed around TH-positive neurons. (c,d) Confocal images of oxDJ-1 in SN dopaminergic neurons and astrocytes. A section containing the SN was immunostained with anti-oxDJ-1 Ab and anti-TH Ab (c) or anti-glial fibrillary acidic protein (GFAP) Ab (d) and then visualized using fluorescence confocal microscopy. A scale bar is shown in each figure. (e) Western blot analyses of oxDJ-1 in separated brain tissues. Protein lysates of each brain site were subjected to western blot analyses using Abs against DJ-1, oxDJ-1, TH, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative band densities of oxDJ-1 normalized to DJ-1 were calculated and are presented as mean ± SD (n = 6). * P

Techniques Used: Mouse Assay, Western Blot, Immunostaining, Staining, Fluorescence, Confocal Microscopy

7) Product Images from "Src regulates insulin secretion and glucose metabolism by influencing subcellular localization of glucokinase in pancreatic β‐cells"

Article Title: Src regulates insulin secretion and glucose metabolism by influencing subcellular localization of glucokinase in pancreatic β‐cells

Journal: Journal of Diabetes Investigation

doi: 10.1111/jdi.12407

Expression levels and subcellular localization of glucokinase. (a) Effects of Src downregulation on expression level of glucokinase messenger ribonucleic acid (mRNA). Expression level of glucokinase was evaluated with semiquantitative real‐time polymerase chain reaction. Data were normalized using β‐actin mRNA ( n = 5 in each group). Values are expressed as mean ± standard error of the mean. (b) Effects of Src downregulation on expression level of glucokinase protein. Lysates of whole INS‐1 cells were used for immunoblotting analysis with antibodies against glucokinase. Quantification data were obtained from four independent experiments and normalized with β‐actin level. Values are expressed as mean ± standard error of the mean. (c) Effects of Src downregulation on subcellular localization of glucokinase. Lysates of whole INS‐1 cells were separated to soluble fraction and pellet. Both fractions were used for immunoblotting analysis with antibodies against glucokinase. Quantification data for each fraction were obtained from four independent experiments and normalized with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) levels for soluble fraction and calnexin levels for pellet. Values are expressed as mean ± standard error of the mean. * P
Figure Legend Snippet: Expression levels and subcellular localization of glucokinase. (a) Effects of Src downregulation on expression level of glucokinase messenger ribonucleic acid (mRNA). Expression level of glucokinase was evaluated with semiquantitative real‐time polymerase chain reaction. Data were normalized using β‐actin mRNA ( n = 5 in each group). Values are expressed as mean ± standard error of the mean. (b) Effects of Src downregulation on expression level of glucokinase protein. Lysates of whole INS‐1 cells were used for immunoblotting analysis with antibodies against glucokinase. Quantification data were obtained from four independent experiments and normalized with β‐actin level. Values are expressed as mean ± standard error of the mean. (c) Effects of Src downregulation on subcellular localization of glucokinase. Lysates of whole INS‐1 cells were separated to soluble fraction and pellet. Both fractions were used for immunoblotting analysis with antibodies against glucokinase. Quantification data for each fraction were obtained from four independent experiments and normalized with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) levels for soluble fraction and calnexin levels for pellet. Values are expressed as mean ± standard error of the mean. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

8) Product Images from "Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies"

Article Title: Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies

Journal: RNA Biology

doi: 10.1080/15476286.2018.1556084

The p53 synthesis efficiency under different stress conditions in NIH3T3 cells. The cells were exposed for 16 hours to doxorubicin (0.1 μM), tunicamycin (1.2 μM) and thapsigargin (0.1 μM), or to an equivalent volume of DMSO, and then harvested. Endogenous p53 protein level was determined by western blot (a) or flow cytometry (b) using monoclonal 1C12 antibody. GAPDH was used as a loading control. The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) was also conducted. For RT-PCR analysis, the samples were prepared using total RNA extracted from mouse fibroblasts treated with DMSO as a control, tunicamycin, thapsigargin and doxorubicin.
Figure Legend Snippet: The p53 synthesis efficiency under different stress conditions in NIH3T3 cells. The cells were exposed for 16 hours to doxorubicin (0.1 μM), tunicamycin (1.2 μM) and thapsigargin (0.1 μM), or to an equivalent volume of DMSO, and then harvested. Endogenous p53 protein level was determined by western blot (a) or flow cytometry (b) using monoclonal 1C12 antibody. GAPDH was used as a loading control. The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) was also conducted. For RT-PCR analysis, the samples were prepared using total RNA extracted from mouse fibroblasts treated with DMSO as a control, tunicamycin, thapsigargin and doxorubicin.

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

9) Product Images from "Minichromosome Maintenance 2 Bound with Retroviral Gp70 Is Localized to Cytoplasm and Enhances DNA-Damage-Induced Apoptosis"

Article Title: Minichromosome Maintenance 2 Bound with Retroviral Gp70 Is Localized to Cytoplasm and Enhances DNA-Damage-Induced Apoptosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040129

Schematic illustration of the structure of MCM2 and its functions in the cytoplasm and nucleus. ( A ) The various functional domains of the MCM2 protein are shown, and the domains and regions required for the activities are indicated. ( B ) Schematic of the novel role of MCM2 in apoptosis enhancement. Normally, MCM2 is recruited into the nucleus for participation in DNA replication. As a result, cellular proliferation is upregulated (proliferation signal). However, when gp70 is present in the cytoplasm, it binds to MCM2 and inhibits its nuclear entry. Furthermore, cytoplasmic gp70-MCM2-complex interacts with PP2A and inhibits its interaction with DNA-PK. Consequently, hyperphosphorylated DNA-PK enhances DNA-damage-induced apoptosis via a P53-related pathway (apoptosis signal).
Figure Legend Snippet: Schematic illustration of the structure of MCM2 and its functions in the cytoplasm and nucleus. ( A ) The various functional domains of the MCM2 protein are shown, and the domains and regions required for the activities are indicated. ( B ) Schematic of the novel role of MCM2 in apoptosis enhancement. Normally, MCM2 is recruited into the nucleus for participation in DNA replication. As a result, cellular proliferation is upregulated (proliferation signal). However, when gp70 is present in the cytoplasm, it binds to MCM2 and inhibits its nuclear entry. Furthermore, cytoplasmic gp70-MCM2-complex interacts with PP2A and inhibits its interaction with DNA-PK. Consequently, hyperphosphorylated DNA-PK enhances DNA-damage-induced apoptosis via a P53-related pathway (apoptosis signal).

Techniques Used: Functional Assay

10) Product Images from "Caffeine Suppresses Apoptosis of Bladder Cancer RT4 Cells in Response to Ionizing Radiation by Inhibiting Ataxia Telangiectasia Mutated-Chk2-p53 Axis"

Article Title: Caffeine Suppresses Apoptosis of Bladder Cancer RT4 Cells in Response to Ionizing Radiation by Inhibiting Ataxia Telangiectasia Mutated-Chk2-p53 Axis

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.168065

Western blotting showing dose-dependent and time-dependent effects of caffeine on γH2AX expression. (a) RT4 cells were pretreated with vehicle or various concentrations of caffeine, as indicated, for 2 h and then treated with 4-Gy ionizing radiation. After 1 h of ionizing radiation, cells were harvested for Western blotting. (b) RT4 cells were pretreated with 0.2 mmol/L caffeine or vehicle for 2 h and then treated with mock or 4-Gy ionizing radiation. Cell lysates were extracted at different time points after exposure, as indicated, and analyzed by Western blotting. (c) Cells were pretreated with vehicle or 0.2 mmol/L caffeine for 2 h, followed by 4-Gy ionizing radiation, and then stained with 53BP1 at 1 h after exposure. Scale bar 20 μm. (d) After pretreatment with vehicle or 0.2 mmol/L caffeine for 2 h and subsequent treatment with 4-Gy ionizing radiation, the relative messenger RNA expressions of p53 and p53 target genes were determined by quantitative real-time polymerase chain reaction after 24 h exposure. All values represent the mean ± standard deviation of three biological repeats in quadruplicate. (e) RT4 cells were pretreated with 20 μmol/L KU55933 or vehicle for 1 h and treated with 4-Gy ionizing radiation or mock as indicated. Cell lysates were extracted at 12 h and 24 h for Western blotting.
Figure Legend Snippet: Western blotting showing dose-dependent and time-dependent effects of caffeine on γH2AX expression. (a) RT4 cells were pretreated with vehicle or various concentrations of caffeine, as indicated, for 2 h and then treated with 4-Gy ionizing radiation. After 1 h of ionizing radiation, cells were harvested for Western blotting. (b) RT4 cells were pretreated with 0.2 mmol/L caffeine or vehicle for 2 h and then treated with mock or 4-Gy ionizing radiation. Cell lysates were extracted at different time points after exposure, as indicated, and analyzed by Western blotting. (c) Cells were pretreated with vehicle or 0.2 mmol/L caffeine for 2 h, followed by 4-Gy ionizing radiation, and then stained with 53BP1 at 1 h after exposure. Scale bar 20 μm. (d) After pretreatment with vehicle or 0.2 mmol/L caffeine for 2 h and subsequent treatment with 4-Gy ionizing radiation, the relative messenger RNA expressions of p53 and p53 target genes were determined by quantitative real-time polymerase chain reaction after 24 h exposure. All values represent the mean ± standard deviation of three biological repeats in quadruplicate. (e) RT4 cells were pretreated with 20 μmol/L KU55933 or vehicle for 1 h and treated with 4-Gy ionizing radiation or mock as indicated. Cell lysates were extracted at 12 h and 24 h for Western blotting.

Techniques Used: Western Blot, Expressing, Staining, Real-time Polymerase Chain Reaction, Standard Deviation

Related Articles

In Vitro:

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Protease Inhibitor:

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Western Blot:

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Immunodetection:

Article Title: Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9
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Blocking Assay:

Article Title: Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection
Article Snippet: .. After blocking with 5% skimmed milk in TBST buffer at 37°C for 30 min, the PVDF membrane was incubated with 1:1,000 diluted RG-M18 mAB in 5% skimmed milk at 37°C for 1 h. The membrane was then washed, and subjected to secondary antibody hybridization with polyclonal goat anti-mouse immunoglobulin/AP at a dilution of 1:5,000 in TBST buffer for 1 h at 37°C. .. After washing, the PVDF membrane was developed under BCIP/NBT substrate solution in the dark for 15 min, and the images were captured using a UVP BioSpectrum 600 Image System.

Purification:

Article Title: Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9
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Electrophoresis:

Article Title: Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies
Article Snippet: Total cell lysates were incubated for 5 min at 95°C and then loaded on a 15% SDS-PAGE gel and following electrophoresis the proteins were transferred onto a nitrocellulose membrane. .. The blot was probed with mouse monoclonal antibodies against p53 (1C12; Cell Signaling), and GAPDH (Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5ʹ, Merck Millipore).

Concentration Assay:

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Incubation:

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other:

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Derivative Assay:

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Polyacrylamide Gel Electrophoresis:

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Lysis:

Article Title: Characterisation of Neutropenia-Associated Neutrophil Elastase Mutations in a Murine Differentiation Model In Vitro and In Vivo
Article Snippet: HEK 293FT cells were washed with PBS, harvested by trypsinisation, extracted in cell lysis buffer (NEB, Cell Signalling, Danvers, MA, USA) supplemented with complete protease inhibitor cocktail (Roche) on ice and boiled in Laemmli buffer at 95°C for 5 min. Hoxb8 cells were washed with PBS and directly lysed in 1x Laemmli buffer (3x 106 cells/50μl) and boiled at 95°C for 5 min. .. 9521, raised against a peptide mapping at the C-terminus of murine NE), BIP (NEB Cell Signaling, Danvers, MA, USA), PDI (NEB Cell Signaling) or GAPDH (Merck Millipore, Darmstadt, Germany).

Staining:

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Article Snippet: .. The other gel was electroblotted onto PVDF membrane (Merck Millipore, Darmstadt, Germany) for 1 h at 200 mA, after which the membrane was stained with NBT solution (0.6 mg/mL NBT, 2 M potassium glycinate at pH 10) in the dark for 45 min. ..

Chloramphenicol Acetyltransferase Assay:

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Hybridization:

Article Title: Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection
Article Snippet: .. After blocking with 5% skimmed milk in TBST buffer at 37°C for 30 min, the PVDF membrane was incubated with 1:1,000 diluted RG-M18 mAB in 5% skimmed milk at 37°C for 1 h. The membrane was then washed, and subjected to secondary antibody hybridization with polyclonal goat anti-mouse immunoglobulin/AP at a dilution of 1:5,000 in TBST buffer for 1 h at 37°C. .. After washing, the PVDF membrane was developed under BCIP/NBT substrate solution in the dark for 15 min, and the images were captured using a UVP BioSpectrum 600 Image System.

Immunofluorescence:

Article Title: v-Src Causes Chromosome Bridges in a Caffeine-Sensitive Manner by Generating DNA Damage
Article Snippet: Antibodies We used rabbit monoclonal anti-phospho-histone H2A.X (Ser139) (γH2AX) (1:400; 2577S, Cell Signaling Technology, Danvers, MA, USA) and mouse monoclonal anti-phosphotyrosine (1:400; clone 4G10, Merck Millipore, Darmstadt, Germany) primary antibodies for immunofluorescence analyses. .. The following primary antibodies were used for immunoblotting: rabbit monoclonal anti-phospho-histone H2A.X (Ser139) (γH2AX) (1:500; 2577S, Cell Signaling Technology) and anti-phospho-Chk1 (Ser345) (1:1000; 133D3, Cell Signaling Technology) antibodies, rabbit polyclonal anti-phospho-KAP1 (Ser824) (1:2000; A300-767A, Bethyl Laboratories, Montgomery, TX, USA), anti-phospho-Chk2 (Thr68) (1:1000; 2661S, Cell Signaling Technology), anti-G3PDH (1:2000; 2275-PC-100, Trevigen), and anti-phospho-Src (Tyr416) (1:2000; #2101, Cell Signaling Technology) antibodies, mouse monoclonal anti-actin (1:2000; clone AC-40, Sigma-Aldrich), anti-Chk1 (1:800; G-4, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Src (1:200; GD11, Millipore), anti-Chk2 (1:1000; clone DCS-273, Medical and Biological Laboratories, Nagoya, Japan), and anti-phosphotyrosine (1:400; clone 4G10, Merck Millipore) antibodies.

SDS Page:

Article Title: Characterisation of Neutropenia-Associated Neutrophil Elastase Mutations in a Murine Differentiation Model In Vitro and In Vivo
Article Snippet: Cell extracts were separated by SDS-PAGE and proteins were transferred onto PVDF membranes. .. 9521, raised against a peptide mapping at the C-terminus of murine NE), BIP (NEB Cell Signaling, Danvers, MA, USA), PDI (NEB Cell Signaling) or GAPDH (Merck Millipore, Darmstadt, Germany).

Article Title: Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies
Article Snippet: Total cell lysates were incubated for 5 min at 95°C and then loaded on a 15% SDS-PAGE gel and following electrophoresis the proteins were transferred onto a nitrocellulose membrane. .. The blot was probed with mouse monoclonal antibodies against p53 (1C12; Cell Signaling), and GAPDH (Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5ʹ, Merck Millipore).

Article Title: Plant Polyphenols Inhibit Functional Amyloid and Biofilm Formation in Pseudomonas Strains by Directing Monomers to Off-Pathway Oligomers
Article Snippet: Detection of Polyphenols by Nitro Blue Tetrazolium (NBT-Assay) FapC monomer incubated with EGCG at different time points were loaded on two 12% SDS-PAGE gel with prestained protein ladder (Thermo Scientific, Roskilde, Denmark). .. The other gel was electroblotted onto PVDF membrane (Merck Millipore, Darmstadt, Germany) for 1 h at 200 mA, after which the membrane was stained with NBT solution (0.6 mg/mL NBT, 2 M potassium glycinate at pH 10) in the dark for 45 min.

Software:

Article Title: Profiling the proteomic inflammatory state of human astrocytes using DIA mass spectrometry
Article Snippet: Immunoblot assays were performed using an anti-human antibody against NFKB1 p105/p50 (Cell Signaling Technology), NFKB2 p100/p52 (Biolegend), and GAPDH (Merck Millipore). .. Band intensities were retrieved using MYImageAnalysis software (Thermo Scientific) and analyzed using Prism software (GraphPad).

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    Merck KGaA mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody
    Expression levels and subcellular localization of glucokinase. (a) Effects of Src downregulation on expression level of glucokinase messenger ribonucleic acid (mRNA). Expression level of glucokinase was evaluated with semiquantitative real‐time polymerase chain reaction. Data were normalized using β‐actin mRNA ( n = 5 in each group). Values are expressed as mean ± standard error of the mean. (b) Effects of Src downregulation on expression level of glucokinase protein. Lysates of whole INS‐1 cells were used for immunoblotting analysis with antibodies against glucokinase. Quantification data were obtained from four independent experiments and normalized with β‐actin level. Values are expressed as mean ± standard error of the mean. (c) Effects of Src downregulation on subcellular localization of glucokinase. Lysates of whole INS‐1 cells were separated to soluble fraction and pellet. Both fractions were used for immunoblotting analysis with antibodies against glucokinase. Quantification data for each fraction were obtained from four independent experiments and normalized with glyceraldehyde <t>3‐phosphate</t> dehydrogenase <t>(GAPDH)</t> levels for soluble fraction and calnexin levels for pellet. Values are expressed as mean ± standard error of the mean. * P
    Mouse Monoclonal Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh Antibody, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody/product/Merck KGaA
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody - by Bioz Stars, 2020-04
    91/100 stars
      Buy from Supplier

    95
    Merck KGaA mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase protein
    Expression levels and subcellular localization of glucokinase. (a) Effects of Src downregulation on expression level of glucokinase messenger ribonucleic acid (mRNA). Expression level of glucokinase was evaluated with semiquantitative real‐time polymerase chain reaction. Data were normalized using β‐actin mRNA ( n = 5 in each group). Values are expressed as mean ± standard error of the mean. (b) Effects of Src downregulation on expression level of glucokinase protein. Lysates of whole INS‐1 cells were used for immunoblotting analysis with antibodies against glucokinase. Quantification data were obtained from four independent experiments and normalized with β‐actin level. Values are expressed as mean ± standard error of the mean. (c) Effects of Src downregulation on subcellular localization of glucokinase. Lysates of whole INS‐1 cells were separated to soluble fraction and pellet. Both fractions were used for immunoblotting analysis with antibodies against glucokinase. Quantification data for each fraction were obtained from four independent experiments and normalized with glyceraldehyde <t>3‐phosphate</t> dehydrogenase <t>(GAPDH)</t> levels for soluble fraction and calnexin levels for pellet. Values are expressed as mean ± standard error of the mean. * P
    Mouse Monoclonal Anti Glyceraldehyde 3 Phosphate Dehydrogenase Protein, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase protein/product/Merck KGaA
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase protein - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

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    Expression levels and subcellular localization of glucokinase. (a) Effects of Src downregulation on expression level of glucokinase messenger ribonucleic acid (mRNA). Expression level of glucokinase was evaluated with semiquantitative real‐time polymerase chain reaction. Data were normalized using β‐actin mRNA ( n = 5 in each group). Values are expressed as mean ± standard error of the mean. (b) Effects of Src downregulation on expression level of glucokinase protein. Lysates of whole INS‐1 cells were used for immunoblotting analysis with antibodies against glucokinase. Quantification data were obtained from four independent experiments and normalized with β‐actin level. Values are expressed as mean ± standard error of the mean. (c) Effects of Src downregulation on subcellular localization of glucokinase. Lysates of whole INS‐1 cells were separated to soluble fraction and pellet. Both fractions were used for immunoblotting analysis with antibodies against glucokinase. Quantification data for each fraction were obtained from four independent experiments and normalized with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) levels for soluble fraction and calnexin levels for pellet. Values are expressed as mean ± standard error of the mean. * P

    Journal: Journal of Diabetes Investigation

    Article Title: Src regulates insulin secretion and glucose metabolism by influencing subcellular localization of glucokinase in pancreatic β‐cells

    doi: 10.1111/jdi.12407

    Figure Lengend Snippet: Expression levels and subcellular localization of glucokinase. (a) Effects of Src downregulation on expression level of glucokinase messenger ribonucleic acid (mRNA). Expression level of glucokinase was evaluated with semiquantitative real‐time polymerase chain reaction. Data were normalized using β‐actin mRNA ( n = 5 in each group). Values are expressed as mean ± standard error of the mean. (b) Effects of Src downregulation on expression level of glucokinase protein. Lysates of whole INS‐1 cells were used for immunoblotting analysis with antibodies against glucokinase. Quantification data were obtained from four independent experiments and normalized with β‐actin level. Values are expressed as mean ± standard error of the mean. (c) Effects of Src downregulation on subcellular localization of glucokinase. Lysates of whole INS‐1 cells were separated to soluble fraction and pellet. Both fractions were used for immunoblotting analysis with antibodies against glucokinase. Quantification data for each fraction were obtained from four independent experiments and normalized with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) levels for soluble fraction and calnexin levels for pellet. Values are expressed as mean ± standard error of the mean. * P

    Article Snippet: Primary antibodies used were mouse monoclonal anti‐Src antibody from Merck KGaA (Darmstadt, Germany); mouse monoclonal anti‐complex I (39 kDa subunit), anti‐complex III (core II), anti‐complex IV (subunit I) and anti‐complex V (subunit α) of mitochondrial respiratory chain antibody from Invitrogen (Eugene, OR, USA); rabbit anti‐glucokinase antibody and anti‐GLUT2 antibody from Abcam (Cambridge, UK); rabbit anti‐Lyn, anti‐Lck, anti‐Fgr, anti‐Blk, anti‐C‐terminal Src kinase (Csk), anti‐calnexin and goat anti‐Hck antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit anti‐neuronal nitric oxide synthase (nNOS) antibody from Cell Signaling Technology (Danvers, MA, USA); mouse monoclonal anti‐β‐actin antibody from Sigma‐Aldrich; and mouse monoclonal anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody from Merck KGaA.

    Techniques: Expressing, Real-time Polymerase Chain Reaction