mouse anti glyceraldehyde 3 phosphate dehydrogenase  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Millipore mouse anti glyceraldehyde 3 phosphate dehydrogenase
    Flotillin expression is downregulated and changes distribution in BeWo cells following foskolin-induced fusion. (A) Representative immunoblots of BeWo cells treated with 20 μM forskolin from 0–72 h. Note that the immunoreactivity of FLOT1 and FLOT2 decreases with forskolin treatment, whereas that of dysferlin (DYSF) increases. <t>Glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in three separate experiments. (B, C) Mononuclear BeWo cells immunolabeled with antibodies against FLOT1 (HPA001393; red in B) and FLOT2 (1608; red in C), respectively. Double arrows indicate areas of flotillin labeling along cellular boundaries; wide arrows denote areas of periuclear staining in individual cells. (D, E) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using antibodies generated against E-cadherin (E-cad, green) and either FLOT1 (red in D) or FLOT2 (red in E), as above. Wide arrows indicate flotillin-like immunoreactivity in crescent-shaped structures in close proximity to clusters of nuclei; arrowhead in E denotes an area of Ecad labeling that has become irregular following cell-cell fusion. (F) Cryosection (5–6 μm) of term placental villus co-immunolabeled using antibodies generated against dysferlin (DYSF, green) and FLOT2 (1608; red) and imaged using confocal microscopy. Single arrows indicate DYSF labeling at the apical regions of the ST; double arrows indicate FLOT2 labeling. (G) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using anti-DYSF (green) and anti-FLOT2 (red) antibodies. Arrowheads denote co-labeling in crescent-shaped structures around clusters of nuclei; the asterisk indicates DYSF immunoreactivity in fused cells. Note that non-fused cells lack DYSF labeling. All scale bars = 20 μm
    Mouse Anti Glyceraldehyde 3 Phosphate Dehydrogenase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti glyceraldehyde 3 phosphate dehydrogenase/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse anti glyceraldehyde 3 phosphate dehydrogenase - by Bioz Stars, 2020-02
    99/100 stars

    Images

    1) Product Images from "Expression of flotillins in the human placenta: potential implications for placental transcytosis"

    Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis

    Journal: Histochemistry and cell biology

    doi: 10.1007/s00418-012-1040-2

    Flotillin expression is downregulated and changes distribution in BeWo cells following foskolin-induced fusion. (A) Representative immunoblots of BeWo cells treated with 20 μM forskolin from 0–72 h. Note that the immunoreactivity of FLOT1 and FLOT2 decreases with forskolin treatment, whereas that of dysferlin (DYSF) increases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in three separate experiments. (B, C) Mononuclear BeWo cells immunolabeled with antibodies against FLOT1 (HPA001393; red in B) and FLOT2 (1608; red in C), respectively. Double arrows indicate areas of flotillin labeling along cellular boundaries; wide arrows denote areas of periuclear staining in individual cells. (D, E) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using antibodies generated against E-cadherin (E-cad, green) and either FLOT1 (red in D) or FLOT2 (red in E), as above. Wide arrows indicate flotillin-like immunoreactivity in crescent-shaped structures in close proximity to clusters of nuclei; arrowhead in E denotes an area of Ecad labeling that has become irregular following cell-cell fusion. (F) Cryosection (5–6 μm) of term placental villus co-immunolabeled using antibodies generated against dysferlin (DYSF, green) and FLOT2 (1608; red) and imaged using confocal microscopy. Single arrows indicate DYSF labeling at the apical regions of the ST; double arrows indicate FLOT2 labeling. (G) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using anti-DYSF (green) and anti-FLOT2 (red) antibodies. Arrowheads denote co-labeling in crescent-shaped structures around clusters of nuclei; the asterisk indicates DYSF immunoreactivity in fused cells. Note that non-fused cells lack DYSF labeling. All scale bars = 20 μm
    Figure Legend Snippet: Flotillin expression is downregulated and changes distribution in BeWo cells following foskolin-induced fusion. (A) Representative immunoblots of BeWo cells treated with 20 μM forskolin from 0–72 h. Note that the immunoreactivity of FLOT1 and FLOT2 decreases with forskolin treatment, whereas that of dysferlin (DYSF) increases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in three separate experiments. (B, C) Mononuclear BeWo cells immunolabeled with antibodies against FLOT1 (HPA001393; red in B) and FLOT2 (1608; red in C), respectively. Double arrows indicate areas of flotillin labeling along cellular boundaries; wide arrows denote areas of periuclear staining in individual cells. (D, E) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using antibodies generated against E-cadherin (E-cad, green) and either FLOT1 (red in D) or FLOT2 (red in E), as above. Wide arrows indicate flotillin-like immunoreactivity in crescent-shaped structures in close proximity to clusters of nuclei; arrowhead in E denotes an area of Ecad labeling that has become irregular following cell-cell fusion. (F) Cryosection (5–6 μm) of term placental villus co-immunolabeled using antibodies generated against dysferlin (DYSF, green) and FLOT2 (1608; red) and imaged using confocal microscopy. Single arrows indicate DYSF labeling at the apical regions of the ST; double arrows indicate FLOT2 labeling. (G) BeWo cells treated with forskolin (20 μM for 72 h) and co-immunolabeled using anti-DYSF (green) and anti-FLOT2 (red) antibodies. Arrowheads denote co-labeling in crescent-shaped structures around clusters of nuclei; the asterisk indicates DYSF immunoreactivity in fused cells. Note that non-fused cells lack DYSF labeling. All scale bars = 20 μm

    Techniques Used: Expressing, Western Blot, Immunolabeling, Labeling, Staining, Generated, Confocal Microscopy

    2) Product Images from "Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation"

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071814

    HGF does not mimic MSC-CM mediated cellular effects. ( A ) Determination of HGF protein content in stimulation media by means of ELISA revealing a robust increase upon MSC conditioning. ( B ) No difference regarding CNPase positivity was observed among OPCs grown in α -MEM in the absence or presence of recombinant HGF, whereas MSC-CM reproducibly increased CNPase expression. ( C–E ) Determination of transcript levels by means of quantitative real-time RT-PCR. No significant differences regarding Id2, Id4 and GFAP transcript levels were observed among OPCs grown in α-MEM in the absence or presence of recombinant HGF. MSC-CM treatment significantly reduced transcript levels of all three genes at all time points. ( F–K ) Anti-HGF antibody mediated depletion experiments revealed no effect on astrocyte (GFAP, Id2, Id4) and oligodendroglial/myelin (CGT, MBP, CNPase) gene expression levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene. All data are shown as mean values ± SEM derived from n = 3 experiments for each analysis. t-test (ns: not significant, * P
    Figure Legend Snippet: HGF does not mimic MSC-CM mediated cellular effects. ( A ) Determination of HGF protein content in stimulation media by means of ELISA revealing a robust increase upon MSC conditioning. ( B ) No difference regarding CNPase positivity was observed among OPCs grown in α -MEM in the absence or presence of recombinant HGF, whereas MSC-CM reproducibly increased CNPase expression. ( C–E ) Determination of transcript levels by means of quantitative real-time RT-PCR. No significant differences regarding Id2, Id4 and GFAP transcript levels were observed among OPCs grown in α-MEM in the absence or presence of recombinant HGF. MSC-CM treatment significantly reduced transcript levels of all three genes at all time points. ( F–K ) Anti-HGF antibody mediated depletion experiments revealed no effect on astrocyte (GFAP, Id2, Id4) and oligodendroglial/myelin (CGT, MBP, CNPase) gene expression levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene. All data are shown as mean values ± SEM derived from n = 3 experiments for each analysis. t-test (ns: not significant, * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Quantitative RT-PCR, Derivative Assay

    Serum reduced conditioned medium. Determination of transcript levels by means of quantitative real-time RT-PCR. Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 10% FBS ( A,C,E ). Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 1% FBS ( B,D,F ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene; data are shown as mean values ± SEM derived from n = 7 for Id2, Id4 and n = 3 for GFAP ( A,C,E ) and n = 3 ( B,D,F ) experiments. t-test (ns: not significant, * P
    Figure Legend Snippet: Serum reduced conditioned medium. Determination of transcript levels by means of quantitative real-time RT-PCR. Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 10% FBS ( A,C,E ). Investigation of GFAP, Id2 and Id4 gene expression levels in α -MEM versus MSC-CM containing 1% FBS ( B,D,F ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference gene; data are shown as mean values ± SEM derived from n = 7 for Id2, Id4 and n = 3 for GFAP ( A,C,E ) and n = 3 ( B,D,F ) experiments. t-test (ns: not significant, * P

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay

    MSC-CM leads to enhanced early myelin expression. Determination of transcript levels by means of quantitative real-time RT-PCR. ( A ) Upregulation of ceramide galactosyltransferase (CGT) expression was detected after three, six and nine days in culture, ( B ) whereas gene expression levels of CNPase were elevated at every measured time point. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference. ( C ) In addition, an increased percentage of CNPase-expressing OPCs was observed among MSC-CM treated cells as compared to cells grown in α -MEM. Significant differences were detected from day three onwards. ( D–E’’’ ) Representative immunofluorescent stainings of CNPase expressing OPCs at all four-time points of investigation. Data are shown as mean values ± SEM and derive from n = 8 (CGT), n = 8 (CNPase, q-RT-PCR) and n = 4 (CNPase, immunostainings) experiments. t-test (* P
    Figure Legend Snippet: MSC-CM leads to enhanced early myelin expression. Determination of transcript levels by means of quantitative real-time RT-PCR. ( A ) Upregulation of ceramide galactosyltransferase (CGT) expression was detected after three, six and nine days in culture, ( B ) whereas gene expression levels of CNPase were elevated at every measured time point. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as reference. ( C ) In addition, an increased percentage of CNPase-expressing OPCs was observed among MSC-CM treated cells as compared to cells grown in α -MEM. Significant differences were detected from day three onwards. ( D–E’’’ ) Representative immunofluorescent stainings of CNPase expressing OPCs at all four-time points of investigation. Data are shown as mean values ± SEM and derive from n = 8 (CGT), n = 8 (CNPase, q-RT-PCR) and n = 4 (CNPase, immunostainings) experiments. t-test (* P

    Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease model mice and macaques"

    Article Title: Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease model mice and macaques

    Journal: Alzheimer's Research & Therapy

    doi: 10.1186/s13195-018-0370-2

    Comparative immunoblot assessment of sortilin relative to human β-amyloid precursor protein (APP) and phosphorylated tau (p-tau) protein products in transgenic mouse cortical extracts. Transgenic tissue homogenates are from the same age groups of amyloid precursor protein and presenilin 1 double-transgenic (APP/PS1), five familial Alzheimer’s disease mutations transgenic (5×FAD), and triple-transgenic Alzheimer’s disease (3×Tg-AD) mice used in histological studies, with lysates from adult C57BL/6 mice and from human patients with AD serving as controls. a and b Western blot images from one batch-processed set of samples. c1 – c4 Quantitative summaries of the protein levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, expressed as a percentage of GAPDH optical density (o.d.) for the groups ( n = 4/group). Levels of the ~ 100 kDa sortilin band representing the full-length protein are comparable between the groups ( a , c1 ). The ~ 15 kDa sortilin band is not readily seen in all mouse brain lysates, in contrast to the human tissue as positive control ( a , c2 ). The human APP protein bands (~ 100 kDa) detected by the 6E10 antibody are distinct in the lysates from the three transgenic models and human cortex, but not in that of the C57BL/6 control ( b , c3 ). Immunoblotted p-tau products migrate as a smear of bands (20–70 kDa), mostly abundant in the human lysates but clearly present in the 3×Tg-AD samples, with minimal amounts in the C57BL/6, APP/PS1, and 5×FAD samples ( b , c4 ). Hash marks to the right of the immunoblot images indicate the band(s) used for densitometry. Statistical results (Kruskal–Wallis nonparametric test with Dunn’s multiple post hoc comparison) are shown in the bar graphs, with the asterisks indicating significant intergroup differences
    Figure Legend Snippet: Comparative immunoblot assessment of sortilin relative to human β-amyloid precursor protein (APP) and phosphorylated tau (p-tau) protein products in transgenic mouse cortical extracts. Transgenic tissue homogenates are from the same age groups of amyloid precursor protein and presenilin 1 double-transgenic (APP/PS1), five familial Alzheimer’s disease mutations transgenic (5×FAD), and triple-transgenic Alzheimer’s disease (3×Tg-AD) mice used in histological studies, with lysates from adult C57BL/6 mice and from human patients with AD serving as controls. a and b Western blot images from one batch-processed set of samples. c1 – c4 Quantitative summaries of the protein levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, expressed as a percentage of GAPDH optical density (o.d.) for the groups ( n = 4/group). Levels of the ~ 100 kDa sortilin band representing the full-length protein are comparable between the groups ( a , c1 ). The ~ 15 kDa sortilin band is not readily seen in all mouse brain lysates, in contrast to the human tissue as positive control ( a , c2 ). The human APP protein bands (~ 100 kDa) detected by the 6E10 antibody are distinct in the lysates from the three transgenic models and human cortex, but not in that of the C57BL/6 control ( b , c3 ). Immunoblotted p-tau products migrate as a smear of bands (20–70 kDa), mostly abundant in the human lysates but clearly present in the 3×Tg-AD samples, with minimal amounts in the C57BL/6, APP/PS1, and 5×FAD samples ( b , c4 ). Hash marks to the right of the immunoblot images indicate the band(s) used for densitometry. Statistical results (Kruskal–Wallis nonparametric test with Dunn’s multiple post hoc comparison) are shown in the bar graphs, with the asterisks indicating significant intergroup differences

    Techniques Used: Transgenic Assay, Mouse Assay, Western Blot, Positive Control

    4) Product Images from "Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells"

    Article Title: Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

    Journal: Skeletal Muscle

    doi: 10.1186/2044-5040-4-5

    Myoblasts require glypican-1 expression for proper hepatocyte growth factor signaling. (A)  Wild-type (WT) C2C12 myoblasts and C6 myoblasts (glypican-1-deficient clone) transiently transfected with rat glypican-1 (C6-Gly), were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. The cell extracts were analyzed by immunoblotting for total HGF receptor (Met) levels, phospho-Met (Tyr 1235/1349), phospho- and total AKT levels, phospho- and total levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2), glypican-1 core protein (after heparitinase treatment), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin. Total Met, AKT and ERK1/2 were used as loading control of its respective phosphorylated forms. GADPH and tubulin were used as loading control of glypican-1 expression levels. The Western blot images are representative of three independent experiments.  (B)  Quantitation of phospho-Met, phospho-AKT and phospho-ERK1/2 from three independent experiments is shown. Values are expressed as mean ± standard deviation. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. * P
    Figure Legend Snippet: Myoblasts require glypican-1 expression for proper hepatocyte growth factor signaling. (A) Wild-type (WT) C2C12 myoblasts and C6 myoblasts (glypican-1-deficient clone) transiently transfected with rat glypican-1 (C6-Gly), were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. The cell extracts were analyzed by immunoblotting for total HGF receptor (Met) levels, phospho-Met (Tyr 1235/1349), phospho- and total AKT levels, phospho- and total levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2), glypican-1 core protein (after heparitinase treatment), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin. Total Met, AKT and ERK1/2 were used as loading control of its respective phosphorylated forms. GADPH and tubulin were used as loading control of glypican-1 expression levels. The Western blot images are representative of three independent experiments. (B) Quantitation of phospho-Met, phospho-AKT and phospho-ERK1/2 from three independent experiments is shown. Values are expressed as mean ± standard deviation. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. * P

    Techniques Used: Expressing, Transfection, Western Blot, Quantitation Assay, Standard Deviation

    5) Product Images from "Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance"

    Article Title: Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance

    Journal: Scientific Reports

    doi: 10.1038/srep20241

    Targeted disruption of the mouse Lrp4 gene. ( a ) Schematic illustration of the Lrp4 gene, targeting vector, and targeted allele. The gray bar indicates the location of a probe for southern blot analysis. Abbreviations: DT-A, diphtheria toxin-A gene; neo, neomycin phosphotransferase gene; B, Bam HI; N, Nco I; X, Xho I. ( b ) Southern blot analysis of genomic DNA from wild-type ( Lrp4 +/+ ), Lrp4 +/− , and Lrp4 −/− mice. Bam HI-digested DNA hybridized with a probe. ( c ) Cell lysate proteins (40 μg protein/lane) from the placenta, foetal membrane, kidneys, and lungs of E18.5 Lrp4 +/+ , Lrp4 +/− , and Lrp4 −/− mice were analyzed by western blot analysis with anti-LRP4 and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. ( d ) Western blot analysis of LRP4 expression in various tissue homogenates from E18.5 foetuses and a maternal mouse. ( e ) LRP4 expression in various cells. C2C12, mouse muscle myoblast; HEPG2, human hepatocellular carcinoma; RAW264.7 and J774A.1, mouse macrophage; P19, mouse embryonal carcinoma. C2C12 cells were differentiated to myotubes by switching to differentiation medium for the indicated days. Two independent primary cultures (1 and 2) of astrocytes were obtained from mouse cerebrum at E18.5. The culture conditions of these cells are described in Supplementary information online . Since two western blot analyses showed similar results, one result was shown. Cropped blots are shown (full-length blots are presented in Supplementary Fig. S6 online ).
    Figure Legend Snippet: Targeted disruption of the mouse Lrp4 gene. ( a ) Schematic illustration of the Lrp4 gene, targeting vector, and targeted allele. The gray bar indicates the location of a probe for southern blot analysis. Abbreviations: DT-A, diphtheria toxin-A gene; neo, neomycin phosphotransferase gene; B, Bam HI; N, Nco I; X, Xho I. ( b ) Southern blot analysis of genomic DNA from wild-type ( Lrp4 +/+ ), Lrp4 +/− , and Lrp4 −/− mice. Bam HI-digested DNA hybridized with a probe. ( c ) Cell lysate proteins (40 μg protein/lane) from the placenta, foetal membrane, kidneys, and lungs of E18.5 Lrp4 +/+ , Lrp4 +/− , and Lrp4 −/− mice were analyzed by western blot analysis with anti-LRP4 and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. ( d ) Western blot analysis of LRP4 expression in various tissue homogenates from E18.5 foetuses and a maternal mouse. ( e ) LRP4 expression in various cells. C2C12, mouse muscle myoblast; HEPG2, human hepatocellular carcinoma; RAW264.7 and J774A.1, mouse macrophage; P19, mouse embryonal carcinoma. C2C12 cells were differentiated to myotubes by switching to differentiation medium for the indicated days. Two independent primary cultures (1 and 2) of astrocytes were obtained from mouse cerebrum at E18.5. The culture conditions of these cells are described in Supplementary information online . Since two western blot analyses showed similar results, one result was shown. Cropped blots are shown (full-length blots are presented in Supplementary Fig. S6 online ).

    Techniques Used: Plasmid Preparation, Southern Blot, Mouse Assay, Western Blot, Expressing

    6) Product Images from "Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease model mice and macaques"

    Article Title: Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease model mice and macaques

    Journal: Alzheimer's Research & Therapy

    doi: 10.1186/s13195-018-0370-2

    Comparative immunoblot assessment of sortilin relative to human β-amyloid precursor protein (APP) and phosphorylated tau (p-tau) protein products in transgenic mouse cortical extracts. Transgenic tissue homogenates are from the same age groups of amyloid precursor protein and presenilin 1 double-transgenic (APP/PS1), five familial Alzheimer’s disease mutations transgenic (5×FAD), and triple-transgenic Alzheimer’s disease (3×Tg-AD) mice used in histological studies, with lysates from adult C57BL/6 mice and from human patients with AD serving as controls. a and b Western blot images from one batch-processed set of samples. c1 – c4 Quantitative summaries of the protein levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, expressed as a percentage of GAPDH optical density (o.d.) for the groups ( n = 4/group). Levels of the ~ 100 kDa sortilin band representing the full-length protein are comparable between the groups ( a , c1 ). The ~ 15 kDa sortilin band is not readily seen in all mouse brain lysates, in contrast to the human tissue as positive control ( a , c2 ). The human APP protein bands (~ 100 kDa) detected by the 6E10 antibody are distinct in the lysates from the three transgenic models and human cortex, but not in that of the C57BL/6 control ( b , c3 ). Immunoblotted p-tau products migrate as a smear of bands (20–70 kDa), mostly abundant in the human lysates but clearly present in the 3×Tg-AD samples, with minimal amounts in the C57BL/6, APP/PS1, and 5×FAD samples ( b , c4 ). Hash marks to the right of the immunoblot images indicate the band(s) used for densitometry. Statistical results (Kruskal–Wallis nonparametric test with Dunn’s multiple post hoc comparison) are shown in the bar graphs, with the asterisks indicating significant intergroup differences
    Figure Legend Snippet: Comparative immunoblot assessment of sortilin relative to human β-amyloid precursor protein (APP) and phosphorylated tau (p-tau) protein products in transgenic mouse cortical extracts. Transgenic tissue homogenates are from the same age groups of amyloid precursor protein and presenilin 1 double-transgenic (APP/PS1), five familial Alzheimer’s disease mutations transgenic (5×FAD), and triple-transgenic Alzheimer’s disease (3×Tg-AD) mice used in histological studies, with lysates from adult C57BL/6 mice and from human patients with AD serving as controls. a and b Western blot images from one batch-processed set of samples. c1 – c4 Quantitative summaries of the protein levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, expressed as a percentage of GAPDH optical density (o.d.) for the groups ( n = 4/group). Levels of the ~ 100 kDa sortilin band representing the full-length protein are comparable between the groups ( a , c1 ). The ~ 15 kDa sortilin band is not readily seen in all mouse brain lysates, in contrast to the human tissue as positive control ( a , c2 ). The human APP protein bands (~ 100 kDa) detected by the 6E10 antibody are distinct in the lysates from the three transgenic models and human cortex, but not in that of the C57BL/6 control ( b , c3 ). Immunoblotted p-tau products migrate as a smear of bands (20–70 kDa), mostly abundant in the human lysates but clearly present in the 3×Tg-AD samples, with minimal amounts in the C57BL/6, APP/PS1, and 5×FAD samples ( b , c4 ). Hash marks to the right of the immunoblot images indicate the band(s) used for densitometry. Statistical results (Kruskal–Wallis nonparametric test with Dunn’s multiple post hoc comparison) are shown in the bar graphs, with the asterisks indicating significant intergroup differences

    Techniques Used: Transgenic Assay, Mouse Assay, Western Blot, Positive Control

    7) Product Images from "Exons 5-15 of Kazrin Are Dispensable for Murine Epidermal Morphogenesis and Homeostasis"

    Article Title: Exons 5-15 of Kazrin Are Dispensable for Murine Epidermal Morphogenesis and Homeostasis

    Journal: The Journal of Investigative Dermatology

    doi: 10.1038/jid.2012.110

    Analysis of kazrin protein expression.  ( a ) Domain architecture of kazrinA and kazrinE. The N terminus of kazrinE is identical to kazrinA. ( b ) Purification of recombinant human kazrinA overexpressed in  Escherichia coli  as a glutathione- S -transferase (GST) fusion protein. ( c ) Lysates of normal human keratinocytes transfected with scrambled small interfering RNA (siControl) or pooled siRNAs specific for all isoforms of kazrin (siKazrinAll) were blotted with pan-kazrin antibody, rabbit pre-immune serum, or rabbit secondary antibody alone. ( d ) Pan-kazrin antibody crosslinked to protein G agarose beads was used to immunoprecipitate endogenous kazrin from lysates of normal human keratinocytes. ( e ,  f ) Pan-kazrin antibody detection of endogenous kazrin, kazrin β-galactosidase (β-gal) fusion protein, and the 28-kDa fragment encoded by kazrin exons 1–4 in lysates of wild-type (wt) mice or litter-matched gene trap (gt/gt) and conditional knockout (flx/flx) mice. simKazrinAll: wt/wt keratinocytes transfected with two pooled siRNAs (see also  Supplementary Figure S1c  online). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control for  c – f .
    Figure Legend Snippet: Analysis of kazrin protein expression. ( a ) Domain architecture of kazrinA and kazrinE. The N terminus of kazrinE is identical to kazrinA. ( b ) Purification of recombinant human kazrinA overexpressed in Escherichia coli as a glutathione- S -transferase (GST) fusion protein. ( c ) Lysates of normal human keratinocytes transfected with scrambled small interfering RNA (siControl) or pooled siRNAs specific for all isoforms of kazrin (siKazrinAll) were blotted with pan-kazrin antibody, rabbit pre-immune serum, or rabbit secondary antibody alone. ( d ) Pan-kazrin antibody crosslinked to protein G agarose beads was used to immunoprecipitate endogenous kazrin from lysates of normal human keratinocytes. ( e , f ) Pan-kazrin antibody detection of endogenous kazrin, kazrin β-galactosidase (β-gal) fusion protein, and the 28-kDa fragment encoded by kazrin exons 1–4 in lysates of wild-type (wt) mice or litter-matched gene trap (gt/gt) and conditional knockout (flx/flx) mice. simKazrinAll: wt/wt keratinocytes transfected with two pooled siRNAs (see also Supplementary Figure S1c online). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control for c – f .

    Techniques Used: Expressing, Purification, Recombinant, Transfection, Small Interfering RNA, Mouse Assay, Knock-Out

    Generation of kazrin β-galactosidase (β-gal) gene-trap (gt/gt) mouse and conditional knockout (flx/flx) mice.  ( a ) Exon structure of mouse kazrin. Blue box represents insertion of the neomycin resistance gene ( β-geo ) cassette in the kazrin β-gal gt/gt mouse. Red triangles indicate loxP sites for removing exon 5 in the kazrin flx/flx mice. ( b ) Predicted domain architecture of kazrin-β-geo fusion protein in the kazrin β-gal (top) gt/gt mouse and kazrin fragment expressed in the (bottom) kazrin flx/flx mouse. ( c ) RT-PCR amplification of transcripts upstream (exons 2–3) or downstream (exons 4–5) of the gene trap. RNA in each lane is from a single representative litter-matched mouse of the genotype indicated. ( d ) Quantitative RT-PCR of transcripts upstream (exons 2–3) and downstream (exons 6–7) of the gene trap. Top: Expression values relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Each bar shows the mean and standard deviation of at least three mice per genotype. Bottom: Ratio (%) of upstream and downstream transcripts from the top panel. Primer positions upstream and downstream of the  β-geo  cassette are indicated by the orange and green brackets, respectively, in  a . ( e ) Alignment of predicted amino-acid sequences encoded by exons 1 and 2 of human kazrinA and mouse kazrin. ( f ) RT-PCR amplification of exons 1–4, 1–5, 2–4, or 2–5 of mouse kazrinA. Positive (GAPDH) and negative (water) controls are shown.  β-geo , β-galactosidase fused to a neomycin resistance gene; Ex, exon.
    Figure Legend Snippet: Generation of kazrin β-galactosidase (β-gal) gene-trap (gt/gt) mouse and conditional knockout (flx/flx) mice. ( a ) Exon structure of mouse kazrin. Blue box represents insertion of the neomycin resistance gene ( β-geo ) cassette in the kazrin β-gal gt/gt mouse. Red triangles indicate loxP sites for removing exon 5 in the kazrin flx/flx mice. ( b ) Predicted domain architecture of kazrin-β-geo fusion protein in the kazrin β-gal (top) gt/gt mouse and kazrin fragment expressed in the (bottom) kazrin flx/flx mouse. ( c ) RT-PCR amplification of transcripts upstream (exons 2–3) or downstream (exons 4–5) of the gene trap. RNA in each lane is from a single representative litter-matched mouse of the genotype indicated. ( d ) Quantitative RT-PCR of transcripts upstream (exons 2–3) and downstream (exons 6–7) of the gene trap. Top: Expression values relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Each bar shows the mean and standard deviation of at least three mice per genotype. Bottom: Ratio (%) of upstream and downstream transcripts from the top panel. Primer positions upstream and downstream of the β-geo cassette are indicated by the orange and green brackets, respectively, in a . ( e ) Alignment of predicted amino-acid sequences encoded by exons 1 and 2 of human kazrinA and mouse kazrin. ( f ) RT-PCR amplification of exons 1–4, 1–5, 2–4, or 2–5 of mouse kazrinA. Positive (GAPDH) and negative (water) controls are shown. β-geo , β-galactosidase fused to a neomycin resistance gene; Ex, exon.

    Techniques Used: Knock-Out, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Expressing, Standard Deviation

    Related Articles

    Transduction:

    Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis
    Article Snippet: The following antibodies were used for immunoblotting and immunolabeling experiments, as indicated in the text and figure legends: mouse anti-FLOT1 (610820, BD Biosciences), mouse anti-FLOT2 (610383, BD Biosciences), rabbit anti-FLOT1 (HPA001393, Sigma-Aldrich), rabbit anti-FLOT2 (F1680, Sigma-Aldrich), rabbit anti-FLOT2 (F1805, Sigma-Aldrich), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon International), mouse anti-dysferlin (DYSF; Ham1/7B6, Vector Laboratories), mouse monoclonal anti-E-cadherin (E-cad, 610181, BD Biosciences), and mouse anti-serine peptidase inhibitor, Kunitz type 1 (SPINT1, clone C76/18) ( ; ; ). .. Forskolin and MISSION Lentiviral Transduction Particles were obtained from Sigma-Aldrich.

    Clone Assay:

    Article Title: COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo
    Article Snippet: Clones were collected in modified RIPA buffer (25 mM Tris–HCl, pH 7.6, 5 mM EDTA, 150 mM NaCl, 1%Triton X-100, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 1 mM phenylmethylsulfonyl fluoride) with protease inhibitor cocktail and centrifuged at 15,000 rpm for 15 min. .. The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000).

    Centrifugation:

    Article Title: Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
    Article Snippet: After centrifugation (15,000 × g for 5 min at 4°C), the soluble fraction was subjected to further analysis. .. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubated for 2 h with the following primary antibodies: mouse anti-HSP90 (SPA-830; Stressgen Biotechnologies, Inc., San Diego, Calif.), mouse anti-HSP70 (SPA-810; Stressgen), rat anti-HSC70 (SPA-815; Stressgen), mouse anti-HSP60 (SPA-806; Stressgen), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International, Temecula, Calif.), rabbit anti-PKR (D-20; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), rabbit anti-phosphorylated PKR (Calbiochem, La Jolla, Calif.), or rabbit anti-BDV P and N in TTBS at 37°C.

    Immunostaining:

    Article Title: Cyclic AMP regulates formation of mammary epithelial acini in vitro
    Article Snippet: Antibodies and reagents Commercial antibodies include: rabbit anti-BIM, anti-pERK, anti-ERK, and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA), rabbit anti–β-catenin and rat anti–α6-integrin (GoH3; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-GM130 (BD Transduction Laboratories, Lexington, KY), mouse anti–KI-67 (Invitrogen, Carlsbad, CA) and mouse anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, Billerica, MA). .. Secondary antibodies include: horseradish peroxidase–coupled (for enhanced chemiluminescence Western blotting) and Alexa Fluor 488 or 555 (for immunostaining), all from Invitrogen.

    SDS-Gel:

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation
    Article Snippet: .. Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies. .. Signals were visualized using IRDye 680LT donkey anti-mouse and IRDye 800CW donkey anti-mouse antibodies (1∶15000) and an Odyssey infrared imaging system scanner (both LI-COR Biosciences, Lincoln, NE).

    Incubation:

    Article Title: Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
    Article Snippet: .. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubated for 2 h with the following primary antibodies: mouse anti-HSP90 (SPA-830; Stressgen Biotechnologies, Inc., San Diego, Calif.), mouse anti-HSP70 (SPA-810; Stressgen), rat anti-HSC70 (SPA-815; Stressgen), mouse anti-HSP60 (SPA-806; Stressgen), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International, Temecula, Calif.), rabbit anti-PKR (D-20; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), rabbit anti-phosphorylated PKR (Calbiochem, La Jolla, Calif.), or rabbit anti-BDV P and N in TTBS at 37°C. .. After three washes in TTBS, horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, Pa.) were applied for 1 h at 37°C.

    Article Title: Valproic Acid Attenuates Microgliosis in Injured Spinal Cord and Purinergic P2X4 Receptor Expression in Activated Microglia
    Article Snippet: The membrane was probed overnight at 4°C with primary antibodies at an appropriate dilution, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) for 1 hr at room temperature. .. Primary antibodies used for the study were as follows: rabbit anti-GFAP, rabbit anti-P2X4 R, rabbit anti-p38MAPK, rabbit antiphosphop38MAPK (Cell Signaling, Danvers, MA), rabbit anti-Mn-SOD (Assay Designs, Ann Arbor, MI), anti-catalase (Abcam), mouse anti-actin regulatory protein (CAPG; Santa Cruz Biotechnology), mouse anti-β-actin (Santa Cruz Biotechnology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon),

    Expressing:

    Article Title: RA-RAR-? counteracts myelin-dependent inhibition of neurite outgrowth via Lingo-1 repression
    Article Snippet: Rabbit polyclonal Lingo-1 (Millipore), mouse anti–β-actin (Sigma-Aldrich), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Millipore) were used as primary antibodies. .. Quantitation of protein expression was performed by densitometry (Photoshop; Adobe) of the representative bands of the immunoblots and normalized to the respective levels of β-actin.

    BIA-KA:

    Article Title: A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice
    Article Snippet: Protein concentrations were determined using the BCA Protein Assay Reagent kit (Bio-Rad Laboratories). .. Rabbit anti-Yap (1:500; Cell Signaling Technology) and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (1:1000; Sigma) were used as the primary antibodies.

    Article Title: Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling
    Article Snippet: Protein levels were quantified with a Pierce BCA kit (Thermo Fisher Scientific) according to the manufacturer's instructions. .. Primary antibodies included rabbit or mouse anti-ZAP-70 (Cell Signaling, Beverly, MA, USA), rabbit anti-Syk/ZAP-70-P (Cell Signaling), rabbit anti-Lyn (Cell Signaling), rabbit anti-Lyn-P (Epitomics, Burlingame, CA, USA), mouse anti-Lck (Cell Signaling), rabbit anti-Btk (Cell Signaling), rabbit anti-Btk-P (Cell Signaling), rabbit anti-ERK1/2 (Cell Signaling), rabbit or mouse anti-ERK1/2-P (Cell Signaling), rabbit anti-Akt (Cell Signaling), rabbit anti-Akt Ser-P (Cell Signaling), mouse anti-tyrosine-P (Millipore, Darmstadt, Germany), rabbit anti-Mcl-1 (Cell Signaling), rabbit anti-PARP (Cell Signaling), rabbit anti-cleaved caspase 3 (Cell Signaling), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma), rabbit anti-α -tubulin (Cell Signaling), and rabbit or mouse anti-β- actin (Sigma).

    Article Title: COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo
    Article Snippet: The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000). .. The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000).

    Modification:

    Article Title: Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells
    Article Snippet: Aliquots with equivalent amounts of protein were subjected to SDS-PAGE in 8% polyacrylamide gels, electrophoretically transferred to Immobilon membranes (EMD Millipore) and probed with the following antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (1:2,000) (Chemicon International, Temecula, CA, USA). .. At 48 hours after transfection, cells were serum-starved for 4 hours, then either treated or not treated with 20 ng/ml [125 I]HGF in Dulbecco’s modified Eagle’s medium (DMEM) 0.1% bovine serum albumin (BSA) for 5 minutes.

    Article Title: COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo
    Article Snippet: Clones were collected in modified RIPA buffer (25 mM Tris–HCl, pH 7.6, 5 mM EDTA, 150 mM NaCl, 1%Triton X-100, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 1 mM phenylmethylsulfonyl fluoride) with protease inhibitor cocktail and centrifuged at 15,000 rpm for 15 min. .. The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000).

    Western Blot:

    Article Title: Cell cycle inhibition limits development and maintenance of neuropathic pain following spinal cord injury
    Article Snippet: Paragraph title: 2.8. Western blot analysis ... Primary antibodies included: mouse anti-cyclin D1 (1:500; Thermal); rabbit anti-CDK4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, UAS); rabbit anti-Iba-1 (1:1000; Wako Chemicals), and mouse anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000; Millipore).

    Article Title: Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
    Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubated for 2 h with the following primary antibodies: mouse anti-HSP90 (SPA-830; Stressgen Biotechnologies, Inc., San Diego, Calif.), mouse anti-HSP70 (SPA-810; Stressgen), rat anti-HSC70 (SPA-815; Stressgen), mouse anti-HSP60 (SPA-806; Stressgen), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International, Temecula, Calif.), rabbit anti-PKR (D-20; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), rabbit anti-phosphorylated PKR (Calbiochem, La Jolla, Calif.), or rabbit anti-BDV P and N in TTBS at 37°C. .. The membranes were washed three times in TTBS, incubated in commercial enhanced chemiluminescence reagent (ECL Western blotting kit; Amersham Pharmacia Biotech, Uppsala, Sweden), and exposed to X-ray film.

    Article Title: Valproic Acid Attenuates Microgliosis in Injured Spinal Cord and Purinergic P2X4 Receptor Expression in Activated Microglia
    Article Snippet: Paragraph title: Western Blotting ... Primary antibodies used for the study were as follows: rabbit anti-GFAP, rabbit anti-P2X4 R, rabbit anti-p38MAPK, rabbit antiphosphop38MAPK (Cell Signaling, Danvers, MA), rabbit anti-Mn-SOD (Assay Designs, Ann Arbor, MI), anti-catalase (Abcam), mouse anti-actin regulatory protein (CAPG; Santa Cruz Biotechnology), mouse anti-β-actin (Santa Cruz Biotechnology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon),

    Article Title: Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells
    Article Snippet: Paragraph title: SDS-PAGE, Western blot and coimmunoprecipitation assays ... Aliquots with equivalent amounts of protein were subjected to SDS-PAGE in 8% polyacrylamide gels, electrophoretically transferred to Immobilon membranes (EMD Millipore) and probed with the following antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (1:2,000) (Chemicon International, Temecula, CA, USA).

    Article Title: A microRNA-Hippo pathway that promotes cardiomyocyte proliferation and cardiac regeneration in mice
    Article Snippet: Paragraph title: Western blot analysis ... Rabbit anti-Yap (1:500; Cell Signaling Technology) and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (1:1000; Sigma) were used as the primary antibodies.

    Article Title: Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease model mice and macaques
    Article Snippet: Paragraph title: Western blot analysis ... Separated proteins were electrotransferred onto Trans-Blot pure nitrocellulose membranes (Bio-Rad Laboratories) and then immunoblotted with rabbit anti-sortilin (1:2000), mouse anti-Aβ 6E10 (1:4000), rabbit anti-phosphorylated tau (T6819, 1:2000), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000; Millipore Shanghai Trading Company Ltd., Shanghai, China) as loading controls.

    Article Title: Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling
    Article Snippet: Paragraph title: Western blotting and immunoprecipitation ... Primary antibodies included rabbit or mouse anti-ZAP-70 (Cell Signaling, Beverly, MA, USA), rabbit anti-Syk/ZAP-70-P (Cell Signaling), rabbit anti-Lyn (Cell Signaling), rabbit anti-Lyn-P (Epitomics, Burlingame, CA, USA), mouse anti-Lck (Cell Signaling), rabbit anti-Btk (Cell Signaling), rabbit anti-Btk-P (Cell Signaling), rabbit anti-ERK1/2 (Cell Signaling), rabbit or mouse anti-ERK1/2-P (Cell Signaling), rabbit anti-Akt (Cell Signaling), rabbit anti-Akt Ser-P (Cell Signaling), mouse anti-tyrosine-P (Millipore, Darmstadt, Germany), rabbit anti-Mcl-1 (Cell Signaling), rabbit anti-PARP (Cell Signaling), rabbit anti-cleaved caspase 3 (Cell Signaling), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma), rabbit anti-α -tubulin (Cell Signaling), and rabbit or mouse anti-β- actin (Sigma).

    Article Title: RA-RAR-? counteracts myelin-dependent inhibition of neurite outgrowth via Lingo-1 repression
    Article Snippet: Rabbit polyclonal Lingo-1 (Millipore), mouse anti–β-actin (Sigma-Aldrich), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Millipore) were used as primary antibodies. .. Quantitation of protein expression was performed by densitometry (Photoshop; Adobe) of the representative bands of the immunoblots and normalized to the respective levels of β-actin.

    Article Title: COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo
    Article Snippet: Paragraph title: Western blotting ... The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000).

    Article Title: Cyclic AMP regulates formation of mammary epithelial acini in vitro
    Article Snippet: Antibodies and reagents Commercial antibodies include: rabbit anti-BIM, anti-pERK, anti-ERK, and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA), rabbit anti–β-catenin and rat anti–α6-integrin (GoH3; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-GM130 (BD Transduction Laboratories, Lexington, KY), mouse anti–KI-67 (Invitrogen, Carlsbad, CA) and mouse anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, Billerica, MA). .. Secondary antibodies include: horseradish peroxidase–coupled (for enhanced chemiluminescence Western blotting) and Alexa Fluor 488 or 555 (for immunostaining), all from Invitrogen.

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation
    Article Snippet: .. Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies. .. Signals were visualized using IRDye 680LT donkey anti-mouse and IRDye 800CW donkey anti-mouse antibodies (1∶15000) and an Odyssey infrared imaging system scanner (both LI-COR Biosciences, Lincoln, NE).

    Transfection:

    Article Title: Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells
    Article Snippet: Aliquots with equivalent amounts of protein were subjected to SDS-PAGE in 8% polyacrylamide gels, electrophoretically transferred to Immobilon membranes (EMD Millipore) and probed with the following antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (1:2,000) (Chemicon International, Temecula, CA, USA). .. For coimmunoprecipitation experiments, wild-type and glypican-1-deficient myoblasts (C6) were transiently transfected as indicated in the figure legends.

    Protease Inhibitor:

    Article Title: Valproic Acid Attenuates Microgliosis in Injured Spinal Cord and Purinergic P2X4 Receptor Expression in Activated Microglia
    Article Snippet: Tissue blocks were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 5% NP-40, and protease inhibitor cocktail (Sigma). .. Primary antibodies used for the study were as follows: rabbit anti-GFAP, rabbit anti-P2X4 R, rabbit anti-p38MAPK, rabbit antiphosphop38MAPK (Cell Signaling, Danvers, MA), rabbit anti-Mn-SOD (Assay Designs, Ann Arbor, MI), anti-catalase (Abcam), mouse anti-actin regulatory protein (CAPG; Santa Cruz Biotechnology), mouse anti-β-actin (Santa Cruz Biotechnology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon),

    Article Title: Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling
    Article Snippet: Western blotting and immunoprecipitation Cell lysates were collected at the indicated times in 1% NP-40 lysis buffer with complete protease inhibitor tablet (Roche, Basel, Switzerland), 1 mM phenylmethanesulfonylfluoride (PMSF), and 2 mM sodium orthovanadate (New England BioLabs, Ipswich, MA, USA). .. Primary antibodies included rabbit or mouse anti-ZAP-70 (Cell Signaling, Beverly, MA, USA), rabbit anti-Syk/ZAP-70-P (Cell Signaling), rabbit anti-Lyn (Cell Signaling), rabbit anti-Lyn-P (Epitomics, Burlingame, CA, USA), mouse anti-Lck (Cell Signaling), rabbit anti-Btk (Cell Signaling), rabbit anti-Btk-P (Cell Signaling), rabbit anti-ERK1/2 (Cell Signaling), rabbit or mouse anti-ERK1/2-P (Cell Signaling), rabbit anti-Akt (Cell Signaling), rabbit anti-Akt Ser-P (Cell Signaling), mouse anti-tyrosine-P (Millipore, Darmstadt, Germany), rabbit anti-Mcl-1 (Cell Signaling), rabbit anti-PARP (Cell Signaling), rabbit anti-cleaved caspase 3 (Cell Signaling), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma), rabbit anti-α -tubulin (Cell Signaling), and rabbit or mouse anti-β- actin (Sigma).

    Article Title: COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo
    Article Snippet: Clones were collected in modified RIPA buffer (25 mM Tris–HCl, pH 7.6, 5 mM EDTA, 150 mM NaCl, 1%Triton X-100, 0.1% sodium dodecyl sulfate, 1% deoxycholate, 1 mM phenylmethylsulfonyl fluoride) with protease inhibitor cocktail and centrifuged at 15,000 rpm for 15 min. .. The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000).

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation
    Article Snippet: Western Blot Analysis Lysis of control and MSC-CM treated OPCs was carried out on ice with radioimmu-noprecipitation assay buffer (RIPA buffer; Cell Signaling Technology, Danvers, Massachusetts, USA) with addition of Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). .. Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies.

    Immunolabeling:

    Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis
    Article Snippet: .. The following antibodies were used for immunoblotting and immunolabeling experiments, as indicated in the text and figure legends: mouse anti-FLOT1 (610820, BD Biosciences), mouse anti-FLOT2 (610383, BD Biosciences), rabbit anti-FLOT1 (HPA001393, Sigma-Aldrich), rabbit anti-FLOT2 (F1680, Sigma-Aldrich), rabbit anti-FLOT2 (F1805, Sigma-Aldrich), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon International), mouse anti-dysferlin (DYSF; Ham1/7B6, Vector Laboratories), mouse monoclonal anti-E-cadherin (E-cad, 610181, BD Biosciences), and mouse anti-serine peptidase inhibitor, Kunitz type 1 (SPINT1, clone C76/18) ( ; ; ). .. Lucifer yellow carbohydrazide (LY-CH), Alexa Fluor 594-conjugated cholera toxin B subunit (CTB-594), Alexa Fluor-conjugated secondary antibodies, and Prolong antifade mounting reagent were obtained from Molecular Probes/Life Technologies.

    Cell Culture:

    Article Title: Exons 5-15 of Kazrin Are Dispensable for Murine Epidermal Morphogenesis and Homeostasis
    Article Snippet: Immunoblotting and immunoprecipitation Mouse and human primary cultured cells were lysed in 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA and 1% Triton X-100. .. The following antibodies were used (dilutions in brackets): rabbit anti-pan-kazrin (1:500), rabbit anti-peptide antibody to all kazrin isoforms (LS7, 1:500) , rabbit anti-kazrin (ProteinTech Group, Manchester, UK; 11572-1-AP; 1:500), rabbit pre-immune serum (1:500), mouse anti-α-tubulin (Sigma, Gillingham, Dorset, UK; T6199; 1:2000), and mouse-anti-glyceraldehyde 3-phosphate dehydrogenase (Millipore, Watford, UK; MAB374; 1:500).

    Article Title: RA-RAR-? counteracts myelin-dependent inhibition of neurite outgrowth via Lingo-1 repression
    Article Snippet: Immunoblotting For immunoblotting, cultured CGNs were collected at 24 h after plating and were lysed on ice for 30 min in a solution containing 10 mM Tris-HCl, 1% Nonidet 40, 150 mM NaCl, 0.1% SDS, and 1% deoxycholate, pH 7.4, containing protease inhibitors (Complete Mini; Roche). .. Rabbit polyclonal Lingo-1 (Millipore), mouse anti–β-actin (Sigma-Aldrich), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Millipore) were used as primary antibodies.

    Imaging:

    Article Title: Exons 5-15 of Kazrin Are Dispensable for Murine Epidermal Morphogenesis and Homeostasis
    Article Snippet: The following antibodies were used (dilutions in brackets): rabbit anti-pan-kazrin (1:500), rabbit anti-peptide antibody to all kazrin isoforms (LS7, 1:500) , rabbit anti-kazrin (ProteinTech Group, Manchester, UK; 11572-1-AP; 1:500), rabbit pre-immune serum (1:500), mouse anti-α-tubulin (Sigma, Gillingham, Dorset, UK; T6199; 1:2000), and mouse-anti-glyceraldehyde 3-phosphate dehydrogenase (Millipore, Watford, UK; MAB374; 1:500). .. Blots labeled with Li-Cor secondary antibodies were imaged using the Li-Cor Odyssey Infrared Imaging System (Li-Cor Biosciences, Cambridge, UK).

    Article Title: Cell cycle inhibition limits development and maintenance of neuropathic pain following spinal cord injury
    Article Snippet: Primary antibodies included: mouse anti-cyclin D1 (1:500; Thermal); rabbit anti-CDK4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, UAS); rabbit anti-Iba-1 (1:1000; Wako Chemicals), and mouse anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000; Millipore). .. Chemiluminescence was captured on a Kodak Image Station 4000R station (Carestream Health Inc., Rochester, NY) and protein bands were quantified by densitometric analysis using Carestream Molecular Imaging Software (Carestream Health Inc., Rochester, NY).

    Article Title: Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells
    Article Snippet: Aliquots with equivalent amounts of protein were subjected to SDS-PAGE in 8% polyacrylamide gels, electrophoretically transferred to Immobilon membranes (EMD Millipore) and probed with the following antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (1:2,000) (Chemicon International, Temecula, CA, USA). .. All immunoreactions were visualized by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) using a ChemiDoc-It 410 high-resolution imaging system (UVP, Upland, CA, USA).

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation
    Article Snippet: Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies. .. Signals were visualized using IRDye 680LT donkey anti-mouse and IRDye 800CW donkey anti-mouse antibodies (1∶15000) and an Odyssey infrared imaging system scanner (both LI-COR Biosciences, Lincoln, NE).

    Protein Concentration:

    Article Title: Valproic Acid Attenuates Microgliosis in Injured Spinal Cord and Purinergic P2X4 Receptor Expression in Activated Microglia
    Article Snippet: Protein concentration was determined using the Bio-Rad DC kit (Bio-Rad, Hercules, CA). .. Primary antibodies used for the study were as follows: rabbit anti-GFAP, rabbit anti-P2X4 R, rabbit anti-p38MAPK, rabbit antiphosphop38MAPK (Cell Signaling, Danvers, MA), rabbit anti-Mn-SOD (Assay Designs, Ann Arbor, MI), anti-catalase (Abcam), mouse anti-actin regulatory protein (CAPG; Santa Cruz Biotechnology), mouse anti-β-actin (Santa Cruz Biotechnology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon),

    Sonication:

    Article Title: Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease model mice and macaques
    Article Snippet: Tissue samples were homogenized by sonication in Pierce T-PER extraction buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing protease inhibitors (Roche, Indianapolis, IN, USA). .. Separated proteins were electrotransferred onto Trans-Blot pure nitrocellulose membranes (Bio-Rad Laboratories) and then immunoblotted with rabbit anti-sortilin (1:2000), mouse anti-Aβ 6E10 (1:4000), rabbit anti-phosphorylated tau (T6819, 1:2000), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000; Millipore Shanghai Trading Company Ltd., Shanghai, China) as loading controls.

    Nucleic Acid Electrophoresis:

    Article Title: Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
    Article Snippet: Equal amounts of the proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, Mass.). .. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubated for 2 h with the following primary antibodies: mouse anti-HSP90 (SPA-830; Stressgen Biotechnologies, Inc., San Diego, Calif.), mouse anti-HSP70 (SPA-810; Stressgen), rat anti-HSC70 (SPA-815; Stressgen), mouse anti-HSP60 (SPA-806; Stressgen), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International, Temecula, Calif.), rabbit anti-PKR (D-20; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), rabbit anti-phosphorylated PKR (Calbiochem, La Jolla, Calif.), or rabbit anti-BDV P and N in TTBS at 37°C.

    CtB Assay:

    Article Title: Expression of flotillins in the human placenta: potential implications for placental transcytosis
    Article Snippet: The following antibodies were used for immunoblotting and immunolabeling experiments, as indicated in the text and figure legends: mouse anti-FLOT1 (610820, BD Biosciences), mouse anti-FLOT2 (610383, BD Biosciences), rabbit anti-FLOT1 (HPA001393, Sigma-Aldrich), rabbit anti-FLOT2 (F1680, Sigma-Aldrich), rabbit anti-FLOT2 (F1805, Sigma-Aldrich), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon International), mouse anti-dysferlin (DYSF; Ham1/7B6, Vector Laboratories), mouse monoclonal anti-E-cadherin (E-cad, 610181, BD Biosciences), and mouse anti-serine peptidase inhibitor, Kunitz type 1 (SPINT1, clone C76/18) ( ; ; ). .. Lucifer yellow carbohydrazide (LY-CH), Alexa Fluor 594-conjugated cholera toxin B subunit (CTB-594), Alexa Fluor-conjugated secondary antibodies, and Prolong antifade mounting reagent were obtained from Molecular Probes/Life Technologies.

    Labeling:

    Article Title: Exons 5-15 of Kazrin Are Dispensable for Murine Epidermal Morphogenesis and Homeostasis
    Article Snippet: The following antibodies were used (dilutions in brackets): rabbit anti-pan-kazrin (1:500), rabbit anti-peptide antibody to all kazrin isoforms (LS7, 1:500) , rabbit anti-kazrin (ProteinTech Group, Manchester, UK; 11572-1-AP; 1:500), rabbit pre-immune serum (1:500), mouse anti-α-tubulin (Sigma, Gillingham, Dorset, UK; T6199; 1:2000), and mouse-anti-glyceraldehyde 3-phosphate dehydrogenase (Millipore, Watford, UK; MAB374; 1:500). .. Fluorescently labeled Li-Cor or horseradish peroxidase-conjugated secondary antibodies were used according to the manufacturer's instructions.

    Article Title: COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo
    Article Snippet: The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000). .. Labeling was detected with the ECL system (Amersham Biosciences, Pittsburgh, PA, USA).

    Mouse Assay:

    Article Title: Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer’s disease model mice and macaques
    Article Snippet: Frontal cortices were blocked from the frozen whole brains or hemibrains of the mice ( n = 4/strain). .. Separated proteins were electrotransferred onto Trans-Blot pure nitrocellulose membranes (Bio-Rad Laboratories) and then immunoblotted with rabbit anti-sortilin (1:2000), mouse anti-Aβ 6E10 (1:4000), rabbit anti-phosphorylated tau (T6819, 1:2000), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000; Millipore Shanghai Trading Company Ltd., Shanghai, China) as loading controls.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
    Article Snippet: Equal amounts of the proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, Mass.). .. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubated for 2 h with the following primary antibodies: mouse anti-HSP90 (SPA-830; Stressgen Biotechnologies, Inc., San Diego, Calif.), mouse anti-HSP70 (SPA-810; Stressgen), rat anti-HSC70 (SPA-815; Stressgen), mouse anti-HSP60 (SPA-806; Stressgen), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International, Temecula, Calif.), rabbit anti-PKR (D-20; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), rabbit anti-phosphorylated PKR (Calbiochem, La Jolla, Calif.), or rabbit anti-BDV P and N in TTBS at 37°C.

    Lysis:

    Article Title: Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
    Article Snippet: Following stress treatments, the cells were washed with cold PBS and then lysed in ice-cold lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 4 mM EDTA, 10 mM NaF, 2 mM Na2 VO3 , 2 mM phenylmethylsulfonyl fluoride). .. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubated for 2 h with the following primary antibodies: mouse anti-HSP90 (SPA-830; Stressgen Biotechnologies, Inc., San Diego, Calif.), mouse anti-HSP70 (SPA-810; Stressgen), rat anti-HSC70 (SPA-815; Stressgen), mouse anti-HSP60 (SPA-806; Stressgen), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International, Temecula, Calif.), rabbit anti-PKR (D-20; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), rabbit anti-phosphorylated PKR (Calbiochem, La Jolla, Calif.), or rabbit anti-BDV P and N in TTBS at 37°C.

    Article Title: Valproic Acid Attenuates Microgliosis in Injured Spinal Cord and Purinergic P2X4 Receptor Expression in Activated Microglia
    Article Snippet: Tissue blocks were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 5% NP-40, and protease inhibitor cocktail (Sigma). .. Primary antibodies used for the study were as follows: rabbit anti-GFAP, rabbit anti-P2X4 R, rabbit anti-p38MAPK, rabbit antiphosphop38MAPK (Cell Signaling, Danvers, MA), rabbit anti-Mn-SOD (Assay Designs, Ann Arbor, MI), anti-catalase (Abcam), mouse anti-actin regulatory protein (CAPG; Santa Cruz Biotechnology), mouse anti-β-actin (Santa Cruz Biotechnology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon),

    Article Title: Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling
    Article Snippet: Western blotting and immunoprecipitation Cell lysates were collected at the indicated times in 1% NP-40 lysis buffer with complete protease inhibitor tablet (Roche, Basel, Switzerland), 1 mM phenylmethanesulfonylfluoride (PMSF), and 2 mM sodium orthovanadate (New England BioLabs, Ipswich, MA, USA). .. Primary antibodies included rabbit or mouse anti-ZAP-70 (Cell Signaling, Beverly, MA, USA), rabbit anti-Syk/ZAP-70-P (Cell Signaling), rabbit anti-Lyn (Cell Signaling), rabbit anti-Lyn-P (Epitomics, Burlingame, CA, USA), mouse anti-Lck (Cell Signaling), rabbit anti-Btk (Cell Signaling), rabbit anti-Btk-P (Cell Signaling), rabbit anti-ERK1/2 (Cell Signaling), rabbit or mouse anti-ERK1/2-P (Cell Signaling), rabbit anti-Akt (Cell Signaling), rabbit anti-Akt Ser-P (Cell Signaling), mouse anti-tyrosine-P (Millipore, Darmstadt, Germany), rabbit anti-Mcl-1 (Cell Signaling), rabbit anti-PARP (Cell Signaling), rabbit anti-cleaved caspase 3 (Cell Signaling), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma), rabbit anti-α -tubulin (Cell Signaling), and rabbit or mouse anti-β- actin (Sigma).

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation
    Article Snippet: Western Blot Analysis Lysis of control and MSC-CM treated OPCs was carried out on ice with radioimmu-noprecipitation assay buffer (RIPA buffer; Cell Signaling Technology, Danvers, Massachusetts, USA) with addition of Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). .. Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies.

    Purification:

    Article Title: Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance
    Article Snippet: Antisera were pre-cleared on a HiTrap NHS-activated HP column (GE Healthcare) coupled with the Trx-His-S protein and purified on a HiTrap NHS-activated HP column coupled with Trx-His-S-mLRP4C50, which contained amino acids 1856–1905 of the C–terminal region of mouse LRP4 , and the purified antibodies were termed LRP4 C50. .. The goat anti-surfactant protein A (sc-7699) and rabbit anti-AQP1 (sc-20810) were purchased from Santa Cruz Biotechnology, while the mouse anti-glyceraldehyde-3-phosphate dehydrogenase (MAB374) was from Chemicon International, Inc.

    SDS Page:

    Article Title: Exons 5-15 of Kazrin Are Dispensable for Murine Epidermal Morphogenesis and Homeostasis
    Article Snippet: Proteins were eluted in standard sample buffer, separated on 4–12% SDS-PAGE gels, and transferred onto nitrocellulose membranes in NuPAGE transfer buffer (Invitrogen) using standard techniques. .. The following antibodies were used (dilutions in brackets): rabbit anti-pan-kazrin (1:500), rabbit anti-peptide antibody to all kazrin isoforms (LS7, 1:500) , rabbit anti-kazrin (ProteinTech Group, Manchester, UK; 11572-1-AP; 1:500), rabbit pre-immune serum (1:500), mouse anti-α-tubulin (Sigma, Gillingham, Dorset, UK; T6199; 1:2000), and mouse-anti-glyceraldehyde 3-phosphate dehydrogenase (Millipore, Watford, UK; MAB374; 1:500).

    Article Title: Valproic Acid Attenuates Microgliosis in Injured Spinal Cord and Purinergic P2X4 Receptor Expression in Activated Microglia
    Article Snippet: Protein extracts (50 μg/lane) were separated by 10% SDS-PAGE and then transferred to a nitrocellulose filter (Millipore, Billerica, MA). .. Primary antibodies used for the study were as follows: rabbit anti-GFAP, rabbit anti-P2X4 R, rabbit anti-p38MAPK, rabbit antiphosphop38MAPK (Cell Signaling, Danvers, MA), rabbit anti-Mn-SOD (Assay Designs, Ann Arbor, MI), anti-catalase (Abcam), mouse anti-actin regulatory protein (CAPG; Santa Cruz Biotechnology), mouse anti-β-actin (Santa Cruz Biotechnology), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Chemicon),

    Article Title: Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells
    Article Snippet: .. Aliquots with equivalent amounts of protein were subjected to SDS-PAGE in 8% polyacrylamide gels, electrophoretically transferred to Immobilon membranes (EMD Millipore) and probed with the following antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (1:2,000) (Chemicon International, Temecula, CA, USA). .. All immunoreactions were visualized by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) using a ChemiDoc-It 410 high-resolution imaging system (UVP, Upland, CA, USA).

    Software:

    Article Title: Cell cycle inhibition limits development and maintenance of neuropathic pain following spinal cord injury
    Article Snippet: Primary antibodies included: mouse anti-cyclin D1 (1:500; Thermal); rabbit anti-CDK4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, UAS); rabbit anti-Iba-1 (1:1000; Wako Chemicals), and mouse anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000; Millipore). .. Chemiluminescence was captured on a Kodak Image Station 4000R station (Carestream Health Inc., Rochester, NY) and protein bands were quantified by densitometric analysis using Carestream Molecular Imaging Software (Carestream Health Inc., Rochester, NY).

    Article Title: Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
    Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then incubated for 2 h with the following primary antibodies: mouse anti-HSP90 (SPA-830; Stressgen Biotechnologies, Inc., San Diego, Calif.), mouse anti-HSP70 (SPA-810; Stressgen), rat anti-HSC70 (SPA-815; Stressgen), mouse anti-HSP60 (SPA-806; Stressgen), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International, Temecula, Calif.), rabbit anti-PKR (D-20; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), rabbit anti-phosphorylated PKR (Calbiochem, La Jolla, Calif.), or rabbit anti-BDV P and N in TTBS at 37°C. .. The intensity of each reactive band was quantified with NIH Image software.

    Article Title: COL25A1 triggers and promotes Alzheimer's disease-like pathology in vivo
    Article Snippet: The primary antibodies were mouse anti-synaptophysin (MAB5258,Chemicon; 1:4,000), mouse anti-BACE1 (MAB5308, Chemicon; 1:1,000), mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000,000) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374A, Chemicon; 1:80,000). .. Densitometric software (Kodak ID 3.6) was used to quantify bands relative to the endogenous control (tubulin or GAPDH), and the values were normalized to those of wild-type (WT) controls.

    Electrophoresis:

    Article Title: Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation
    Article Snippet: .. Probes were subjected to standard SDS gel electrophoresis and Western blotting using RunBlue SDS gels (Expedeon, Cambridgeshire, UK), RunBlue Blot Sandwich nitrocellulose (Expedeon) applying mouse anti-MBP (MBP; Covance; 1∶500, Princeton, New Jersey, USA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore; 1∶1000) antibodies. .. Signals were visualized using IRDye 680LT donkey anti-mouse and IRDye 800CW donkey anti-mouse antibodies (1∶15000) and an Odyssey infrared imaging system scanner (both LI-COR Biosciences, Lincoln, NE).

    Radio Immunoprecipitation:

    Article Title: Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells
    Article Snippet: For analysis of phosphorylated proteins, cell extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer in the presence of phosphatase inhibitors as previously described [ , ]. .. Aliquots with equivalent amounts of protein were subjected to SDS-PAGE in 8% polyacrylamide gels, electrophoretically transferred to Immobilon membranes (EMD Millipore) and probed with the following antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti-α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (1:2,000) (Chemicon International, Temecula, CA, USA).

    Quantitation Assay:

    Article Title: RA-RAR-? counteracts myelin-dependent inhibition of neurite outgrowth via Lingo-1 repression
    Article Snippet: Rabbit polyclonal Lingo-1 (Millipore), mouse anti–β-actin (Sigma-Aldrich), and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Millipore) were used as primary antibodies. .. Quantitation of protein expression was performed by densitometry (Photoshop; Adobe) of the representative bands of the immunoblots and normalized to the respective levels of β-actin.

    Immunoprecipitation:

    Article Title: Exons 5-15 of Kazrin Are Dispensable for Murine Epidermal Morphogenesis and Homeostasis
    Article Snippet: Paragraph title: Immunoblotting and immunoprecipitation ... The following antibodies were used (dilutions in brackets): rabbit anti-pan-kazrin (1:500), rabbit anti-peptide antibody to all kazrin isoforms (LS7, 1:500) , rabbit anti-kazrin (ProteinTech Group, Manchester, UK; 11572-1-AP; 1:500), rabbit pre-immune serum (1:500), mouse anti-α-tubulin (Sigma, Gillingham, Dorset, UK; T6199; 1:2000), and mouse-anti-glyceraldehyde 3-phosphate dehydrogenase (Millipore, Watford, UK; MAB374; 1:500).

    Article Title: Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling
    Article Snippet: Paragraph title: Western blotting and immunoprecipitation ... Primary antibodies included rabbit or mouse anti-ZAP-70 (Cell Signaling, Beverly, MA, USA), rabbit anti-Syk/ZAP-70-P (Cell Signaling), rabbit anti-Lyn (Cell Signaling), rabbit anti-Lyn-P (Epitomics, Burlingame, CA, USA), mouse anti-Lck (Cell Signaling), rabbit anti-Btk (Cell Signaling), rabbit anti-Btk-P (Cell Signaling), rabbit anti-ERK1/2 (Cell Signaling), rabbit or mouse anti-ERK1/2-P (Cell Signaling), rabbit anti-Akt (Cell Signaling), rabbit anti-Akt Ser-P (Cell Signaling), mouse anti-tyrosine-P (Millipore, Darmstadt, Germany), rabbit anti-Mcl-1 (Cell Signaling), rabbit anti-PARP (Cell Signaling), rabbit anti-cleaved caspase 3 (Cell Signaling), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma), rabbit anti-α -tubulin (Cell Signaling), and rabbit or mouse anti-β- actin (Sigma).

    Staining:

    Article Title: Cyclic AMP regulates formation of mammary epithelial acini in vitro
    Article Snippet: Antibodies and reagents Commercial antibodies include: rabbit anti-BIM, anti-pERK, anti-ERK, and anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA), rabbit anti–β-catenin and rat anti–α6-integrin (GoH3; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-GM130 (BD Transduction Laboratories, Lexington, KY), mouse anti–KI-67 (Invitrogen, Carlsbad, CA) and mouse anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, Billerica, MA). .. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    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: 90/100, 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 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mouse anti bax monoclonal - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    89
    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: 89/100, 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/Millipore
    Average 89 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, 2020-02
    89/100 stars
      Buy from Supplier

    95
    Millipore anti glyceraldehyde 3 phosphate dehydrogenase
    Na v 1.1 levels are reduced in Scn1a RX/+ mice, and this reduction is not prevented by tau ablation. Levels of Na v 1.1 and total sodium channels (pan Na v ) in the parietal cortex of 8-month-old mice were determined by Western blot analysis. <t>Glyceraldehyde-3-phosphate</t> dehydrogenase (GAPDH) levels were used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype). The average Na v 1.1 to pan Na v ratio in Scn1a +/+ / Tau +/+ mice was arbitrarily defined as 1.0. ***p
    Anti Glyceraldehyde 3 Phosphate Dehydrogenase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti glyceraldehyde 3 phosphate dehydrogenase/product/Millipore
    Average 95 stars, based on 88 article reviews
    Price from $9.99 to $1999.99
    anti glyceraldehyde 3 phosphate dehydrogenase - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    Image Search Results


    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

    Na v 1.1 levels are reduced in Scn1a RX/+ mice, and this reduction is not prevented by tau ablation. Levels of Na v 1.1 and total sodium channels (pan Na v ) in the parietal cortex of 8-month-old mice were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype). The average Na v 1.1 to pan Na v ratio in Scn1a +/+ / Tau +/+ mice was arbitrarily defined as 1.0. ***p

    Journal: Annals of Neurology

    Article Title: Tau Reduction Prevents Disease in a Mouse Model of Dravet Syndrome

    doi: 10.1002/ana.24230

    Figure Lengend Snippet: Na v 1.1 levels are reduced in Scn1a RX/+ mice, and this reduction is not prevented by tau ablation. Levels of Na v 1.1 and total sodium channels (pan Na v ) in the parietal cortex of 8-month-old mice were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype). The average Na v 1.1 to pan Na v ratio in Scn1a +/+ / Tau +/+ mice was arbitrarily defined as 1.0. ***p

    Article Snippet: After blocking for 1 hour in 5% bovine serum albumin diluted in Tris-buffered saline (BSA-TBS), membranes were incubated overnight at 4°C in anti-Nav 1.1 (1:1,000; Alomone Labs, Jerusalem, Israel), anti–pan-sodium channel (Pan Nav, 1:1,000; Sigma), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; Millipore, Billerica, MA), anti-tau clone Tau-5 (1:3,000; Life Technologies), anti-tau clone EP2456Y (1:1,000; Millipore), anti–phospho-tau Ser 396/404 clone PHF-1 (1:3,000, a gift from Dr P. Davies), anti–phospho-tau Thr231 clone CP9 (1:25, a gift from Dr P. Davies), or anti–phospho-PHF-tau pSer202+Thr205 clone AT8 (1:80; Thermo Scientific, Waltham, MA).

    Techniques: Mouse Assay, Western Blot

    Cortical levels of total and phosphorylated tau are not altered in Scn1a RX/+ mice. Levels of phospho-tau (PHF-1, Ser396/Ser404; AT8, Ser202/Thr205; CP9, Thr231) and total tau (Tau-5, EP2456Y) in the parietal cortex of 8-month-old mice of the indicated genotypes were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype) revealed no statistically significant differences between Scn1a RX/+ /Tau +/+ and Scn1a +/+ / Tau +/+ mice (Student t test). Average phospho-tau to EP2456Y ratios (PHF-1, AT8, CP9) or average total tau levels (Tau-5, EP2456Y) in Scn1a +/+ / Tau +/+ mice were arbitrarily defined as 1.0. Values represent mean ± standard error of the mean.

    Journal: Annals of Neurology

    Article Title: Tau Reduction Prevents Disease in a Mouse Model of Dravet Syndrome

    doi: 10.1002/ana.24230

    Figure Lengend Snippet: Cortical levels of total and phosphorylated tau are not altered in Scn1a RX/+ mice. Levels of phospho-tau (PHF-1, Ser396/Ser404; AT8, Ser202/Thr205; CP9, Thr231) and total tau (Tau-5, EP2456Y) in the parietal cortex of 8-month-old mice of the indicated genotypes were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype) revealed no statistically significant differences between Scn1a RX/+ /Tau +/+ and Scn1a +/+ / Tau +/+ mice (Student t test). Average phospho-tau to EP2456Y ratios (PHF-1, AT8, CP9) or average total tau levels (Tau-5, EP2456Y) in Scn1a +/+ / Tau +/+ mice were arbitrarily defined as 1.0. Values represent mean ± standard error of the mean.

    Article Snippet: After blocking for 1 hour in 5% bovine serum albumin diluted in Tris-buffered saline (BSA-TBS), membranes were incubated overnight at 4°C in anti-Nav 1.1 (1:1,000; Alomone Labs, Jerusalem, Israel), anti–pan-sodium channel (Pan Nav, 1:1,000; Sigma), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; Millipore, Billerica, MA), anti-tau clone Tau-5 (1:3,000; Life Technologies), anti-tau clone EP2456Y (1:1,000; Millipore), anti–phospho-tau Ser 396/404 clone PHF-1 (1:3,000, a gift from Dr P. Davies), anti–phospho-tau Thr231 clone CP9 (1:25, a gift from Dr P. Davies), or anti–phospho-PHF-tau pSer202+Thr205 clone AT8 (1:80; Thermo Scientific, Waltham, MA).

    Techniques: Mouse Assay, Western Blot

    Changes in p21 WAF1/CIP1 and p53 in CerS2 null mouse liver. A , Western blot showing changes in p21 WAF1/CIP1 levels. The values show the fold difference in p21 WAF1/CIP1 levels in WT (+/+) versus CerS2 null mice (−/−), normalized to glyceraldehyde-3-phosphate

    Journal: The Journal of Biological Chemistry

    Article Title: A Critical Role for Ceramide Synthase 2 in Liver Homeostasis

    doi: 10.1074/jbc.M109.077610

    Figure Lengend Snippet: Changes in p21 WAF1/CIP1 and p53 in CerS2 null mouse liver. A , Western blot showing changes in p21 WAF1/CIP1 levels. The values show the fold difference in p21 WAF1/CIP1 levels in WT (+/+) versus CerS2 null mice (−/−), normalized to glyceraldehyde-3-phosphate

    Article Snippet: An anti-p21WAF1/CIP1 antibody was from Santa Cruz Biotechnology; anti-actin was from MP Biomedicals; anti-α-tubulin was from Sigma; and anti-glyceraldehyde-3-phosphate dehydrogenase was from Millipore.

    Techniques: Western Blot, Mouse Assay