mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh  (Millipore)


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    Name:
    GAPDH
    Description:
    GAPDH Glyceraldehyde 3 phosphate dehydrogenase catalyzes the conversion of glyceraldehyde 3 phosphate into D glycerate 1 3 bisphosphate as part of the glycolysis pathway GAPDH has also been found to function in additional cellular process such as transcription apoptosis oxidative stress and ER to Golgi transport
    Catalog Number:
    g5262
    Price:
    None
    Applications:
    GAPDH protein is suitable for use as a molecular weight marker and protein standard for molecular biology applications, including western blotting and mass spectometry.
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    Structured Review

    Millipore mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh
    ALS2 loss promotes an accumulation of insoluble proteins in the spinal cord of SOD1 H46R mice. Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH)</t> and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.
    GAPDH Glyceraldehyde 3 phosphate dehydrogenase catalyzes the conversion of glyceraldehyde 3 phosphate into D glycerate 1 3 bisphosphate as part of the glycolysis pathway GAPDH has also been found to function in additional cellular process such as transcription apoptosis oxidative stress and ER to Golgi transport
    https://www.bioz.com/result/mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh/product/Millipore
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh - by Bioz Stars, 2020-05
    99/100 stars

    Images

    1) Product Images from "Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking"

    Article Title: Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009805

    ALS2 loss promotes an accumulation of insoluble proteins in the spinal cord of SOD1 H46R mice. Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.
    Figure Legend Snippet: ALS2 loss promotes an accumulation of insoluble proteins in the spinal cord of SOD1 H46R mice. Western blot analysis of the levels of proteins, including ALS2, SOD1, ubiquitin (Ub), polyubiquitin binding protein p62/SQSTM1 (p62), microtubule-associated protein 1-light chain 3 (LC3), peripherin, neurofilament heavy chain (NFH), TAR DNA-binding protein 43-kD (TDP-43), heat-shock protein Hsp70, 20S proteasome subunits (α5, C2, and LMP2), vimentin, and glial fibrillary acidic protein (GFAP), in the lumbo-sacral cord from 8, 12, 16, and 20 week-old mice with four distinct genotypes; wild-type ( Als2 +/+ ), Als2 +/+ ; SOD1 H46R , Als2 +/− ; SOD1 H46R , and Als2 −/− ; SOD1 H46R . Two fractions; 1% Triton X-soluble fraction (TX-soluble; left panels) and 1% Triton X-insoluble/5% SDS-soluble fraction (TX-insoluble; right panels) were analyzed. SOD1_mono and SOD1_HMW represent monomeric and high molecular-weight (aggregated) forms of SOD1, respectively. Ub_mono and Ub_HMW represent monomeric ubiquitin and the polyubiquitinated proteins, respectively. LC-I and LC-II are cytosolic and lipidated forms of LC3, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin served as controls for TX-soluble and TX-insoluble fractions, respectively.

    Techniques Used: Mouse Assay, Western Blot, Binding Assay, Molecular Weight

    2) Product Images from "Hepatocyte-Derived Lipocalin 2 Is a Potential Serum Biomarker Reflecting Tumor Burden in Hepatoblastoma"

    Article Title: Hepatocyte-Derived Lipocalin 2 Is a Potential Serum Biomarker Reflecting Tumor Burden in Hepatoblastoma

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2018.05.006

    Evaluating inflammatory cytokines and NF-κB signaling in hepatoblastoma (HB) occurring in lipocalin 2 (Lcn2) knockout (KO) versus wild-type (WT) mice after Yes-associated protein-1 (YAP1)–β-catenin co-expression. A: Gene expression of inflammatory cytokines IL-1β, IL-6, and interferon (IFN)-γ was detected using real time-PCR, showing high levels of variability within tumor-laden livers of both WT and Lcn2 KO HBs. No significant difference in cytokine expression is detected in baseline WT and Lcn2 KO livers. B: Lcn2 KO HB shows decreased total levels of p65 and the activated phospho-Ser536 p65 by Western blot. Protein levels were quantified using densitometry. There is no statistically significant difference between WT and Lcn2 KO HBs. C: Gene expression of p65 targets FAS, inducible nitric oxide synthase (iNOS), and Myc was detected using real-time PCR, showing high levels of variability within tumor-laden livers of both WT and Lcn2 KO HBs. No significant difference in gene expression is detected in baseline WT and Lcn2 KO livers. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: Evaluating inflammatory cytokines and NF-κB signaling in hepatoblastoma (HB) occurring in lipocalin 2 (Lcn2) knockout (KO) versus wild-type (WT) mice after Yes-associated protein-1 (YAP1)–β-catenin co-expression. A: Gene expression of inflammatory cytokines IL-1β, IL-6, and interferon (IFN)-γ was detected using real time-PCR, showing high levels of variability within tumor-laden livers of both WT and Lcn2 KO HBs. No significant difference in cytokine expression is detected in baseline WT and Lcn2 KO livers. B: Lcn2 KO HB shows decreased total levels of p65 and the activated phospho-Ser536 p65 by Western blot. Protein levels were quantified using densitometry. There is no statistically significant difference between WT and Lcn2 KO HBs. C: Gene expression of p65 targets FAS, inducible nitric oxide synthase (iNOS), and Myc was detected using real-time PCR, showing high levels of variability within tumor-laden livers of both WT and Lcn2 KO HBs. No significant difference in gene expression is detected in baseline WT and Lcn2 KO livers. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Lipocalin 2 (LCN2) is regulated by Yes-associated protein-1 (YAP1)–β-catenin in hepatoblastomas (HBs). A: Venn diagram of up-regulated genes in a mouse genome analysis illustrating that five gene loci that contain binding sites for T-cell factor 4 (TCF4) and TEA domain (TEAD) also have up-regulated expression in murine HB. B: Real-time PCR of liver lysates from control mouse livers and β-catenin–YAP1–induced HB tumor-laden livers, showing significantly increased expression of LCN2. C: Serum enzyme-linked immunosorbent assay (ELISA) for LCN2 was performed on mice with HB tumors and varying degrees of tumor burden, approximated by liver weight–to–body weight (LW/BW) ratio, showing a positive and significant correlation between serum LCN2 level and disease burden in mice ( R 2 = 0.82). D: Schematic representation of the timeline for HepG2 cell transfection with silencing RNA targeting β-catenin, YAP1 , LCN2 , or scrambled siRNA. E: Western blot shows decreased levels of LCN2 after transfection of HepG2 cells with siRNA to LCN2 , β-catenin, or YAP1 . Efficacy of each of the siRNA is shown by decrease in their respective protein levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ∗∗∗∗ P
    Figure Legend Snippet: Lipocalin 2 (LCN2) is regulated by Yes-associated protein-1 (YAP1)–β-catenin in hepatoblastomas (HBs). A: Venn diagram of up-regulated genes in a mouse genome analysis illustrating that five gene loci that contain binding sites for T-cell factor 4 (TCF4) and TEA domain (TEAD) also have up-regulated expression in murine HB. B: Real-time PCR of liver lysates from control mouse livers and β-catenin–YAP1–induced HB tumor-laden livers, showing significantly increased expression of LCN2. C: Serum enzyme-linked immunosorbent assay (ELISA) for LCN2 was performed on mice with HB tumors and varying degrees of tumor burden, approximated by liver weight–to–body weight (LW/BW) ratio, showing a positive and significant correlation between serum LCN2 level and disease burden in mice ( R 2 = 0.82). D: Schematic representation of the timeline for HepG2 cell transfection with silencing RNA targeting β-catenin, YAP1 , LCN2 , or scrambled siRNA. E: Western blot shows decreased levels of LCN2 after transfection of HepG2 cells with siRNA to LCN2 , β-catenin, or YAP1 . Efficacy of each of the siRNA is shown by decrease in their respective protein levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ∗∗∗∗ P

    Techniques Used: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Mouse Assay, Transfection, Western Blot

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    Electrophoresis:

    Article Title: Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer
    Article Snippet: .. After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes and reacted with primary antibodies against P53 (Imaxgen), P21 (Eptomics) and GAPDH (Sigma). .. After incubation, membranes were washed and incubated with anti-rabbit secondary antibodies (R & D, USA).

    Immunohistochemistry:

    Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
    Article Snippet: .. Antibodies The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. Proximity ligation assay (PLA) To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

    Co-Immunoprecipitation Assay:

    Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
    Article Snippet: .. Antibodies The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. Proximity ligation assay (PLA) To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

    Incubation:

    Article Title: IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells
    Article Snippet: .. Proteins were transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes and blocked with 5% BSA in Tween-TBS for 1 h The membranes were then incubated (4°C) in 5% BSA in Tween-TBS with antibodies (1:1000) against phospho-AMPK-Thr172, total AMPK, phospho-Akt-Thr308, phospho-Akt substrate, total Akt, phospho-Jak1, total Jak1, phospho-Jak3, total Jak3, phospho-STAT3-Tyr705, total STAT3, phospho-STAT5, total STAT5 (Cell Signaling), GLUT4 (Santa Cruz), and GAPDH (Sigma). ..

    Proximity Ligation Assay:

    Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
    Article Snippet: .. Antibodies The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. Proximity ligation assay (PLA) To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

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    Millipore mouse anti bax monoclonal
    Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of <t>pro-caspase-8/9/3</t> (A) and <t>Bcl-2/Bax</t> (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P
    Mouse Anti Bax Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti bax monoclonal/product/Millipore
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse anti bax monoclonal - by Bioz Stars, 2020-05
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    99
    Millipore mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody
    Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to <t>glyceraldehyde-3-phosphate</t> dehydrogenase <t>(GAPDH)</t> at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.
    Mouse Monoclonal Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 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/Millipore
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody - by Bioz Stars, 2020-05
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    Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Expressing, Cell Culture, Transfection, Western Blot, Positive Control

    Effects of mGluR4 activation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two mGluR4-specific siRNAs (simGluR4-1 and simGluR4-2) for 24 h, followed by treatment with the vehicle (Ctrl) or 30 μM of VU0155041 for 24 h. Then, the differential expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) , 9 (D) , 3 (E) , Bcl-2 (F) , Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) , each statistical value represents the mean ± SD of at least three independent experiments. * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Effects of mGluR4 activation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two mGluR4-specific siRNAs (simGluR4-1 and simGluR4-2) for 24 h, followed by treatment with the vehicle (Ctrl) or 30 μM of VU0155041 for 24 h. Then, the differential expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) , 9 (D) , 3 (E) , Bcl-2 (F) , Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) , each statistical value represents the mean ± SD of at least three independent experiments. * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Activation Assay, Expressing, Cell Culture, Transfection, Western Blot

    Gli-1 downregulation is involved in mGluR4-mediated dynamic expression of apoptosis-related proteins in LN229 cells. (A,B) LN229 cells were treated with the vehicle (Ctrl), 5 μg/mL shh, or 30 μM VU0155041 plus 5 μg/mL shh (VU + shh) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments. * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Gli-1 downregulation is involved in mGluR4-mediated dynamic expression of apoptosis-related proteins in LN229 cells. (A,B) LN229 cells were treated with the vehicle (Ctrl), 5 μg/mL shh, or 30 μM VU0155041 plus 5 μg/mL shh (VU + shh) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments. * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Effects of Gli-1 downregulation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two Gli-1-specific siRNAs (siGli-1-1 and siGli-1-2) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. Shh protein (5 μg/mL) was simultaneously added to the non-transfected LN229 cells as a positive control. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments ( n = 5). * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Expressing, Cell Culture, Transfection, Western Blot, Positive Control

    Effects of mGluR4 activation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two mGluR4-specific siRNAs (simGluR4-1 and simGluR4-2) for 24 h, followed by treatment with the vehicle (Ctrl) or 30 μM of VU0155041 for 24 h. Then, the differential expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) , 9 (D) , 3 (E) , Bcl-2 (F) , Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) , each statistical value represents the mean ± SD of at least three independent experiments. * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Effects of mGluR4 activation on the expression of apoptosis-related proteins in LN229 cells. (A,B) Cultured LN229 cells were transfected with non-specific siRNA (siNC) and two mGluR4-specific siRNAs (simGluR4-1 and simGluR4-2) for 24 h, followed by treatment with the vehicle (Ctrl) or 30 μM of VU0155041 for 24 h. Then, the differential expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) , 9 (D) , 3 (E) , Bcl-2 (F) , Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) , each statistical value represents the mean ± SD of at least three independent experiments. * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Activation Assay, Expressing, Cell Culture, Transfection, Western Blot

    Gli-1 downregulation is involved in mGluR4-mediated dynamic expression of apoptosis-related proteins in LN229 cells. (A,B) LN229 cells were treated with the vehicle (Ctrl), 5 μg/mL shh, or 30 μM VU0155041 plus 5 μg/mL shh (VU + shh) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments. * P

    Journal: Frontiers in Neuroscience

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells

    doi: 10.3389/fnins.2018.00320

    Figure Lengend Snippet: Gli-1 downregulation is involved in mGluR4-mediated dynamic expression of apoptosis-related proteins in LN229 cells. (A,B) LN229 cells were treated with the vehicle (Ctrl), 5 μg/mL shh, or 30 μM VU0155041 plus 5 μg/mL shh (VU + shh) for 24 h. Then, the expression of pro-caspase-8/9/3 (A) and Bcl-2/Bax (B) was determined by western blot (WB) analysis. (C–H) WB bands were quantified to generate the ratios of pro-caspase-8 (C) ,−9 (D) , and−3 (E) , Bcl-2 (F) , and Bax (G) to β-actin and the ratio of Bcl-2 to Bax (H) ; each statistical value represents the mean ± SD of three independent experiments. * P

    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Techniques: Expressing, Western Blot

    Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.

    Journal: Genes & Development

    Article Title: Foxp1 regulation of neonatal vocalizations via cortical development

    doi: 10.1101/gad.305037.117

    Figure Lengend Snippet: Sumoylation of FOXP1. ( A ) Representative immunoblotting of Foxp1 in the mouse neocortex during development. ( Right panel) Quantification of SUMO–Foxp1 immunoblotting. Immunoblots were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at each time point and then subsequently normalized to nonsumoylated Foxp1 levels at embryonic day 15.5 (E15.5). Data are represented as means (±SEM). n = 3 per condition. ( B ) Endogenous coimmunoprecipitation of Foxp1 and SUMO-1 in the mouse neocortex at P0. ( C ) Schematic of FOXP1 protein showing the location of K636. (PolyQ) Polyglutamine motif; (ZF) zinc finger; (LZ) leucine zipper. ( D ) K636 is conserved across species. ( E ) Immunoblotting for Flag-tagged FOXP1 in 293T cells. Lysates were treated with 1 mM H 2 O 2 for 1 h ( left panel) or 100 µM ginkgolic acid for 6 h ( right panel) in the presence or absence of NEM. (FOXP1 WT) Flag-tagged wild-type FOXP1. The asterisk indicates a nonspecific band of the SUMO-1 antibody. ( F ) Immunoblot of immunoprecipitated wild-type Flag-tagged FOXP1 or Flag-tagged FOXP1 KR.

    Article Snippet: The following antibodies were used: mouse monoclonal anti-SUMO-1 (D-11) antibody (Santa Cruz Biotechnology, sc-5308), rabbit polyclonal anti-FOXP1 antibody , mouse monoclonal anti-FOXP1 (JC12) antibody (Abcam, ab32010), goat polyclonal anti-FOXP2 (N-16) antibody (Santa Cruz Biotechnology, sc-21068), mouse monoclonal anti-Flag M2 antibody (Sigma-Aldrich, F1804), mouse monoclonal anti-V5 antibody (Invitrogen, R960-25), goat polyclonal anti-GFP antibody (Rockland Immunochemicals, 600-101-215), chick polyclonal anti-GFP antibody (Aves Laboratories, GFP-1010), rabbit monoclonal anti-SUMO-2/3 (18H8) antibody (Cell Signaling Technology, 4971), rabbit polyclonal anti-PIAS2 antibody (Abcam, ab155556), rabbit polyclonal anti-PIAS3 (H-169) antibody (Santa Cruz Biotechnology, sc-14017), rabbit polyclonal anti-MAP2 antibody (Chemicon, AB5622), mouse monoclonal anti-CtBP (E-12) antibody (Santa Cruz Biotechnology, sc-17759), rabbit polyclonal anti-CDP (CUX1: M-222) antibody (Santa Cruz Biotechnology, sc-13024), rat anti-CTIP2 (Abcam, ab18465), rabbit polyclonal anti-HDAC1 antibody (Abcam, ab19845), mouse monoclonal anti-HDAC1 (10E2) antibody (Cell Signaling Technology, 5256), mouse monoclonal anti-HDAC2 (3F3) antibody (Cell Signaling Technology, 5113), rabbit monoclonal anti-MTA1 (D40D1) XP antibody (Cell Signaling Technology, 5647), rabbit polyclonal anti-MTA2 (H-170) antibody (Santa Cruz Biotechnology, sc-28731), rabbit polyclonal anti-p66β (GATAD2B) antibody (Novus Biologicals, NBP1-87358), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Millipore, MAB374), mouse (G3A1) mAb IgG1 isotype control (Cell Signaling Technology, 5415), normal rabbit IgG (Cell Signaling Technology, 2729), and normal goat IgG (Santa Cruz Biotechnology, sc-2028).

    Techniques: Western Blot, Immunoprecipitation

    Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: A Buffered Alcohol-Based Fixative for Histomorphologic and Molecular Applications

    doi: 10.1369/0022155416649579

    Figure Lengend Snippet: Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.

    Article Snippet: The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST; 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) for 1 hr, washed, and incubated overnight at 4C in TBST with rabbit anti-AQP1 polyclonal antibody (Cat. No. sc-20810; dilution 1:100; Santa Cruz Biotechnology, Dallas, TA) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5; dilution 1:3000; Calbiochem, Gibbstown, NJ).

    Techniques: BIA-KA, Protein Extraction, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page