anti gapdh  (Millipore)


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    Anti GAPDH
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    Structured Review

    Millipore anti gapdh
    Anti GAPDH

    https://www.bioz.com/result/anti gapdh/product/Millipore
    Average 99 stars, based on 1300 article reviews
    Price from $9.99 to $1999.99
    anti gapdh - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma"

    Article Title: Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11299

    Expression of Kv1.3 potassium channel in different pancreatic ductal adenocarcinoma cell lines A . Histogram of the distribution of Kcna3 gene expression in a panel of pancreatic cancer cell lines from Affymetrix U133 Gene Chip. Arbitrary intensity units of the probe ID (207237_at) for Kcna3; the intensity values were normalized with dCHIP2006 software ( www.dchip.org ) B . The relative level of gene expression as determined by quantitative RT-PCR was calculated using qBase_Biogazelle software, which allows a multiple reference genes-normalization and performs inter-run calibration. Actb and Ywhaz of non-tumoral HPDE cells were set as reference genes (value 1) to normalize the gene expression. C . Whole cell extracts (50 μg/lane) from different PDAC cell lines were loaded on SDS-PAGE. Western blot revealed Kv1.3 bands (multiple bands are presumably due to glycosylation according to manufacturer or to degradation products) at around 65 kDa. These bands correlated with Kv1.3 expression since none of the bands were present in a cell line silenced for Kv1.3 (not shown). The same blot was developed with the antibody against GAPDH (45 kDa) as loading control. D . Whole cell extract (Tot ext), enriched membranous fraction (Memb) and Percoll-purified mitochondria (mitos) fractions obtained from Colo357 were loaded at equal protein concentration (40 μg/lane). Enrichment in the mitochondrial marker Bak and decrease of the intensity of SERCA (ER marker) and PMCA (PM marker) indicates a higher purity of the mitochondrial preparation. Results in C-D are representative of Western blots from three independent experiments.
    Figure Legend Snippet: Expression of Kv1.3 potassium channel in different pancreatic ductal adenocarcinoma cell lines A . Histogram of the distribution of Kcna3 gene expression in a panel of pancreatic cancer cell lines from Affymetrix U133 Gene Chip. Arbitrary intensity units of the probe ID (207237_at) for Kcna3; the intensity values were normalized with dCHIP2006 software ( www.dchip.org ) B . The relative level of gene expression as determined by quantitative RT-PCR was calculated using qBase_Biogazelle software, which allows a multiple reference genes-normalization and performs inter-run calibration. Actb and Ywhaz of non-tumoral HPDE cells were set as reference genes (value 1) to normalize the gene expression. C . Whole cell extracts (50 μg/lane) from different PDAC cell lines were loaded on SDS-PAGE. Western blot revealed Kv1.3 bands (multiple bands are presumably due to glycosylation according to manufacturer or to degradation products) at around 65 kDa. These bands correlated with Kv1.3 expression since none of the bands were present in a cell line silenced for Kv1.3 (not shown). The same blot was developed with the antibody against GAPDH (45 kDa) as loading control. D . Whole cell extract (Tot ext), enriched membranous fraction (Memb) and Percoll-purified mitochondria (mitos) fractions obtained from Colo357 were loaded at equal protein concentration (40 μg/lane). Enrichment in the mitochondrial marker Bak and decrease of the intensity of SERCA (ER marker) and PMCA (PM marker) indicates a higher purity of the mitochondrial preparation. Results in C-D are representative of Western blots from three independent experiments.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Software, Quantitative RT-PCR, SDS Page, Western Blot, Purification, Protein Concentration, Marker

    2) Product Images from "cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1"

    Article Title: cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1

    Journal: Journal of Molecular and Cellular Cardiology

    doi: 10.1016/j.yjmcc.2014.11.012

    CCN1 promotes VSMC proliferation, migration and chemotaxis . Western blot analysis of CCN1 and GAPDH protein 24 h post-transfection with siNEG (□) or siCCN1(∎) (A). Relative BrDU incorporation 24–40 h post-transfection with siNEG of siCCN1; n = 3 (B). Barrier migration assay of siNEG (□) and siCCN1(∎) transfected cells after 18 h stimulation with 2.5 ng/ml or 10 ng/ml PDGF BB ; n = 6 (C and D). Boyden-chamber chemotaxis assay using 5 or 20 μg/ml recombinant CCN1 in the bottom chamber for 8 h; n = 6 (E and F). □ indicates controls; ∎ indicates stimulated cells. * indicates p
    Figure Legend Snippet: CCN1 promotes VSMC proliferation, migration and chemotaxis . Western blot analysis of CCN1 and GAPDH protein 24 h post-transfection with siNEG (□) or siCCN1(∎) (A). Relative BrDU incorporation 24–40 h post-transfection with siNEG of siCCN1; n = 3 (B). Barrier migration assay of siNEG (□) and siCCN1(∎) transfected cells after 18 h stimulation with 2.5 ng/ml or 10 ng/ml PDGF BB ; n = 6 (C and D). Boyden-chamber chemotaxis assay using 5 or 20 μg/ml recombinant CCN1 in the bottom chamber for 8 h; n = 6 (E and F). □ indicates controls; ∎ indicates stimulated cells. * indicates p

    Techniques Used: Migration, Chemotaxis Assay, Western Blot, Transfection, BrdU Incorporation Assay, Recombinant

    3) Product Images from "Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells"

    Article Title: Histone Deacetylase 1 and 3 Regulate the Mesodermal Lineage Commitment of Mouse Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113262

    TSA induces early differentiation of ESCs and promotes mesodermal lineage differentiation. ( A ) Bright-field images, alkaline phosphatase staining of ESCs and representative immunofluorescence images of Oct4 staining in control or TSA-treated ESCs (10 and 20 ng/ml) in the presence of LIF. ( B ) Western blotting verification of H3, acetyl-H3, H4, and acetyl-H4 in control or TSA-treated ESCs (10 and 20 ng/ml). GAPDH was used as a loading control. ( C ) The relative expression levels of Oct4, Nanog, and Rex1 mRNA in control or TSA-treated ESCs (10 and 20 ng/ml). ( D, E ) QRT-PCR analysis for marker genes of three germ layers (endoderm, mesoderm and ectoderm) in control or TSA-treated ESCs (10 and 20 ng/ml), under the monolayer differentiation condition without LIF. The cells were treated by TSA after removing LIF for 24h and collected mRNA for QRT-PCR analysis at day 3 of monolayer differentiation. ( F, G ) QRT-PCR analysis for marker genes of the three germ layers in control or TSA-treated ESCs (10 and 20 ng/ml) during EB differentiation. The EBs was treated by TSA from day 2 to 6 of EB differentiation. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: TSA induces early differentiation of ESCs and promotes mesodermal lineage differentiation. ( A ) Bright-field images, alkaline phosphatase staining of ESCs and representative immunofluorescence images of Oct4 staining in control or TSA-treated ESCs (10 and 20 ng/ml) in the presence of LIF. ( B ) Western blotting verification of H3, acetyl-H3, H4, and acetyl-H4 in control or TSA-treated ESCs (10 and 20 ng/ml). GAPDH was used as a loading control. ( C ) The relative expression levels of Oct4, Nanog, and Rex1 mRNA in control or TSA-treated ESCs (10 and 20 ng/ml). ( D, E ) QRT-PCR analysis for marker genes of three germ layers (endoderm, mesoderm and ectoderm) in control or TSA-treated ESCs (10 and 20 ng/ml), under the monolayer differentiation condition without LIF. The cells were treated by TSA after removing LIF for 24h and collected mRNA for QRT-PCR analysis at day 3 of monolayer differentiation. ( F, G ) QRT-PCR analysis for marker genes of the three germ layers in control or TSA-treated ESCs (10 and 20 ng/ml) during EB differentiation. The EBs was treated by TSA from day 2 to 6 of EB differentiation. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Staining, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Marker, Two Tailed Test

    Loss of HDAC1 or 3 enhances mesodermal lineage differentiation. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in shHDAC1 and shHDAC3 ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the knockdown of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis of mesoderm genes in shHDAC1 ESCs and control cells at the days 0, 3, 6, and 10 during EB differentiation. ( D ) QRT-PCR analysis of mesoderm genes in shHDAC3 ESCs and control cells during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, shHDAC1, and shHDAC3 cells after 9 days of EB formation. Green, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: Loss of HDAC1 or 3 enhances mesodermal lineage differentiation. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in shHDAC1 and shHDAC3 ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the knockdown of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis of mesoderm genes in shHDAC1 ESCs and control cells at the days 0, 3, 6, and 10 during EB differentiation. ( D ) QRT-PCR analysis of mesoderm genes in shHDAC3 ESCs and control cells during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, shHDAC1, and shHDAC3 cells after 9 days of EB formation. Green, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Staining, Western Blot, Quantitative RT-PCR, Immunofluorescence, Expressing, Two Tailed Test

    The histone deacetylase activity of HDACs is required for the regulation of mesoderm gene. ( A ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC1-OE (H1 OE), and TSA-treated H1-OE cells. GAPDH was used as a loading control. ( B, C ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H1-OE, and TSA-treated H1-OE cells. ( D ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC3-OE (H1 OE), and TSA-treated H3-OE cells. GAPDH was used as a loading control. ( E, F ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H3-OE, and TSA-treated H3-OE cells. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: The histone deacetylase activity of HDACs is required for the regulation of mesoderm gene. ( A ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC1-OE (H1 OE), and TSA-treated H1-OE cells. GAPDH was used as a loading control. ( B, C ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H1-OE, and TSA-treated H1-OE cells. ( D ) Western blotting verification of acetyl-H4 and H4 expression levels in control, HDAC3-OE (H1 OE), and TSA-treated H3-OE cells. GAPDH was used as a loading control. ( E, F ) QRT-PCR analysis of the three germ layer genes at day 6 of EB differentiation in control, H3-OE, and TSA-treated H3-OE cells. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Histone Deacetylase Assay, Activity Assay, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test

    Ectopic expression of HDAC1 and 3 inhibits the differentiation into the mesodermal lineage in EBs. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in control, HDAC1-overexpression (HDAC1-OE), and HDAC3-overexpression (HDAC3-OE) ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the overexpression of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis for the mRNA levels of mesoderm genes in HDAC1-OE ESCs, HDAC3-OE ESCs and control cells during EB differentiation. ( D ) Western blotting analysis of the Gata4 and α-SMA protein levels in HDAC3-OE ESCs and control cell lines during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, HDAC1-OE, and HDAC3-OE cells after 9 days of EB formation. Red, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P
    Figure Legend Snippet: Ectopic expression of HDAC1 and 3 inhibits the differentiation into the mesodermal lineage in EBs. ( A ) Bright-field images and alkaline phosphatase staining of ESCs in control, HDAC1-overexpression (HDAC1-OE), and HDAC3-overexpression (HDAC3-OE) ESCs. ( B ) Western blotting verification and QRT-PCR analysis of the overexpression of HDAC1 and HDAC3 in stable E14 cell lines. GAPDH was used as a loading control. ( C ) QRT-PCR analysis for the mRNA levels of mesoderm genes in HDAC1-OE ESCs, HDAC3-OE ESCs and control cells during EB differentiation. ( D ) Western blotting analysis of the Gata4 and α-SMA protein levels in HDAC3-OE ESCs and control cell lines during EB differentiation. ( E ) Representative immunofluorescence images for the GATA4 expression level in control, HDAC1-OE, and HDAC3-OE cells after 9 days of EB formation. Red, Gata4; blue, Hoechst 33342 for nuclei staining. Data are expressed as means ± SD. Statistical significance was assessed by two-tailed Student's t test. ***, P

    Techniques Used: Expressing, Staining, Over Expression, Western Blot, Quantitative RT-PCR, Immunofluorescence, Two Tailed Test

    4) Product Images from "Secreted Protein Acidic and Rich in Cysteine (SPARC) Enhances Cell Proliferation, Migration, and Epithelial Mesenchymal Transition, and SPARC Expression is Associated with Tumor Grade in Head and Neck Cancer"

    Article Title: Secreted Protein Acidic and Rich in Cysteine (SPARC) Enhances Cell Proliferation, Migration, and Epithelial Mesenchymal Transition, and SPARC Expression is Associated with Tumor Grade in Head and Neck Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18071556

    Investigation of SPARC treatment-induced signaling pathways by Western blot: Detroit 562 cells were treated with SPARC for 1.5 h and the protein extract was subjected to Western blot assay. ( A ) Phosphorylation status of ribosomal s6 kinase1 (RSK1), mitogen and stress-activated protein kinase (MSK) , and extracellular signal-regulated kinase (ERK); ( B ) phosphorylation status of p70S6K, AKT, 4e-BP1 and ( C ) investigation of the effect of ERK, RSK, and AKT inhibitor. Band intensity was normalized to GAPDH and values are expressed relative to the control group.
    Figure Legend Snippet: Investigation of SPARC treatment-induced signaling pathways by Western blot: Detroit 562 cells were treated with SPARC for 1.5 h and the protein extract was subjected to Western blot assay. ( A ) Phosphorylation status of ribosomal s6 kinase1 (RSK1), mitogen and stress-activated protein kinase (MSK) , and extracellular signal-regulated kinase (ERK); ( B ) phosphorylation status of p70S6K, AKT, 4e-BP1 and ( C ) investigation of the effect of ERK, RSK, and AKT inhibitor. Band intensity was normalized to GAPDH and values are expressed relative to the control group.

    Techniques Used: Western Blot

    5) Product Images from "Resveratrol sequentially induces replication and oxidative stresses to drive p53-CXCR2 mediated cellular senescence in cancer cells"

    Article Title: Resveratrol sequentially induces replication and oxidative stresses to drive p53-CXCR2 mediated cellular senescence in cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00315-4

    CXCR2 protects cells from undergoing stress-induced apoptosis. ( A ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured 3 days after treatment with RSV (25 μM) by flow cytometry. ( B ) Cells were treated as in ( A ) and whole-cell extracts were collected for Western blot analysis using BCL2 and BAX antibodies. ( C ) Downregulation of CXCR2 by siRNA as measured by RT-PCR and flow cytometry. U2OS cells were transfected with siRNA duplexes (200 nmol/L) specific to CXCR2 or scrabbled oligo in serum-free medium for 6 hours, then were incubated with complete medium for 24 h and then incubated with RSV for 3 days. ( D ) U2OS cells were treated the same as in ( C ) and apoptosis was measured by flow cytometry. ( E ) The U2OS cells were treated the same as in ( C ) and whole-cell extracts were collected for Western blot analysis using BCL2 antibodies. ( F ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured by flow cytometry 2 days after treatment with H 2 O 2 (400 μM). Results shown are representative of three independent experiments. ( G ) The shNeg and shCXCR2 NHF cells were treated the same as in ( F ) and apoptosis was measured by flow cytometry. The numbers shown below Western blot images are means (first row) and SE (second row) of band intensities relative to control. Signals on the immunoblots were analyzed by ImageJ, normalized with that of GAPDH.
    Figure Legend Snippet: CXCR2 protects cells from undergoing stress-induced apoptosis. ( A ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured 3 days after treatment with RSV (25 μM) by flow cytometry. ( B ) Cells were treated as in ( A ) and whole-cell extracts were collected for Western blot analysis using BCL2 and BAX antibodies. ( C ) Downregulation of CXCR2 by siRNA as measured by RT-PCR and flow cytometry. U2OS cells were transfected with siRNA duplexes (200 nmol/L) specific to CXCR2 or scrabbled oligo in serum-free medium for 6 hours, then were incubated with complete medium for 24 h and then incubated with RSV for 3 days. ( D ) U2OS cells were treated the same as in ( C ) and apoptosis was measured by flow cytometry. ( E ) The U2OS cells were treated the same as in ( C ) and whole-cell extracts were collected for Western blot analysis using BCL2 antibodies. ( F ) Apoptosis in shNeg and shCXCR2 U2OS cells was measured by flow cytometry 2 days after treatment with H 2 O 2 (400 μM). Results shown are representative of three independent experiments. ( G ) The shNeg and shCXCR2 NHF cells were treated the same as in ( F ) and apoptosis was measured by flow cytometry. The numbers shown below Western blot images are means (first row) and SE (second row) of band intensities relative to control. Signals on the immunoblots were analyzed by ImageJ, normalized with that of GAPDH.

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

    6) Product Images from "Human Cytomegalovirus-Induced Degradation of CYTIP Modulates Dendritic Cell Adhesion and Migration"

    Article Title: Human Cytomegalovirus-Induced Degradation of CYTIP Modulates Dendritic Cell Adhesion and Migration

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00461

    Human cytomegalovirus (HCMV) infection of mature dendritic cells (mDCs) leads to decreased CYTIP expression levels . (A) mDCs were either mock- or herpes simplex virus type 1 (HSV-1)-infected and harvested 24 hpi, (B) mock- or HCMV-infected and sorted based on their green fluorescence protein expression 24 hpi. (A,B) Protein lysates were prepared and subjected to Western blot analyses with antibodies specific for CYTIP, cytohesin-1, ICP0, IE2, or GAPDH. One representative experiment out of at least three is shown. (C) Quantification of CYTIP and cytohesin-1 protein expression levels in HSV-1-infected or HCMV-positive mDCs, summarizing at least three independent experiments. Expression of CYTIP and cytohesin-1 was normalized to the GAPDH control. Mock was set to 100%. Significant changes (**** = p
    Figure Legend Snippet: Human cytomegalovirus (HCMV) infection of mature dendritic cells (mDCs) leads to decreased CYTIP expression levels . (A) mDCs were either mock- or herpes simplex virus type 1 (HSV-1)-infected and harvested 24 hpi, (B) mock- or HCMV-infected and sorted based on their green fluorescence protein expression 24 hpi. (A,B) Protein lysates were prepared and subjected to Western blot analyses with antibodies specific for CYTIP, cytohesin-1, ICP0, IE2, or GAPDH. One representative experiment out of at least three is shown. (C) Quantification of CYTIP and cytohesin-1 protein expression levels in HSV-1-infected or HCMV-positive mDCs, summarizing at least three independent experiments. Expression of CYTIP and cytohesin-1 was normalized to the GAPDH control. Mock was set to 100%. Significant changes (**** = p

    Techniques Used: Infection, Expressing, Fluorescence, Western Blot

    7) Product Images from "The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles"

    Article Title: The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/7094781

    Western blot analysis of SOD1, SOD2, SOD3, catalase, and TRX in HUVECs (a–e) and MVs (g). GSH (total content) was analysed using a specific kit (f); the left panel shows the standard curve and the right panel the total GSH content. Y: young; S: senescent. HUVEC protein data were normalized against β -actin (a–d) or GAPDH (e); however, the GSH data (f) were normalized against the protein content of the sample. Protein data for the MVs was normalized against the intensity of red Ponceau staining. Bars represent mean ± SD ( n = 3 pools). ∗ p
    Figure Legend Snippet: Western blot analysis of SOD1, SOD2, SOD3, catalase, and TRX in HUVECs (a–e) and MVs (g). GSH (total content) was analysed using a specific kit (f); the left panel shows the standard curve and the right panel the total GSH content. Y: young; S: senescent. HUVEC protein data were normalized against β -actin (a–d) or GAPDH (e); however, the GSH data (f) were normalized against the protein content of the sample. Protein data for the MVs was normalized against the intensity of red Ponceau staining. Bars represent mean ± SD ( n = 3 pools). ∗ p

    Techniques Used: Western Blot, Staining

    8) Product Images from "Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells"

    Article Title: Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells

    Journal: British Journal of Cancer

    doi: 10.1038/s41416-018-0284-6

    Analysis of predicted upstream regulators via ingenuity pathway analysis and biochemical confirmation. a Scatter plot depicting the predicted activation/inhibition status of upstream regulators belonging to the category of ‘transcription factors’. The x -axis represents the activation z -score, and the y -axis represents the negative logarithm of the significance of overlap between a regulator’s target genes and the differentially expressed genes in Pfn KD vs control samples. Regulators with an overlap p -value ≤0.01, and z -scores ≥2.0 or ≤−2.0 are considered to be significantly activated or inhibited, respectively. b List of target genes for the transcription factors SMAD3 and SPDEF, and their fold-change of expression (hatched bar: direction of gene changes consistent with predicted activation of SMAD3; grey bar: direction of gene changes not consistent; white bar: effects of direction of gene changes unknown). c Immunoblot analyses of SMAD3 and pSMAD3C from 3D BME-MoT extracts of MDA-231 cells transfected with the indicated siRNAs (GAPDH blot—loading control). d Immunoblot analyses of FAK, pFAK, SMAD3 and pSMAD3C from 3D extracts of control- vs Pfn1-shRNA expressing MDA-231 cells cultured in BME with or without collagen-I (data representative from three independent experiments)
    Figure Legend Snippet: Analysis of predicted upstream regulators via ingenuity pathway analysis and biochemical confirmation. a Scatter plot depicting the predicted activation/inhibition status of upstream regulators belonging to the category of ‘transcription factors’. The x -axis represents the activation z -score, and the y -axis represents the negative logarithm of the significance of overlap between a regulator’s target genes and the differentially expressed genes in Pfn KD vs control samples. Regulators with an overlap p -value ≤0.01, and z -scores ≥2.0 or ≤−2.0 are considered to be significantly activated or inhibited, respectively. b List of target genes for the transcription factors SMAD3 and SPDEF, and their fold-change of expression (hatched bar: direction of gene changes consistent with predicted activation of SMAD3; grey bar: direction of gene changes not consistent; white bar: effects of direction of gene changes unknown). c Immunoblot analyses of SMAD3 and pSMAD3C from 3D BME-MoT extracts of MDA-231 cells transfected with the indicated siRNAs (GAPDH blot—loading control). d Immunoblot analyses of FAK, pFAK, SMAD3 and pSMAD3C from 3D extracts of control- vs Pfn1-shRNA expressing MDA-231 cells cultured in BME with or without collagen-I (data representative from three independent experiments)

    Techniques Used: Activation Assay, Inhibition, Expressing, Multiple Displacement Amplification, Transfection, shRNA, Cell Culture

    Effects of perturbation of SMAD3 on 3D outgrowth of MDA-231 cells in BME matrix. a – b a: SMAD3 and GAPDH (loading control) immunoblots of MDA-231 cells transfected with either empty vector (EV) control or SMAD3 overexpression vector (cells were transfected with 1 µg plasmid in 35 mm culture dish). Note that the SMAD3 immunoblot image was acquired at a very low exposure to avoid signal saturation of the overexpression band. b Summarises the outgrowth of SMAD3 overexpression group relative to EV control group of cells in BME matrix. c – d c : SMAD3 and GAPDH (loading control) immunoblots of total cell extracts of control and Pfn1-shRNA expressing MDA-231 cells transiently transfected with the indicated siRNAs. d Summarises the outgrowth of the various transfected groups relative to the control group (control shRNA expressers transfected with control siRNA) in BME matrix. Gene silencing and overexpression-based outgrowth data summarised from two independent experiments, with three technical replicates per experiment). * p
    Figure Legend Snippet: Effects of perturbation of SMAD3 on 3D outgrowth of MDA-231 cells in BME matrix. a – b a: SMAD3 and GAPDH (loading control) immunoblots of MDA-231 cells transfected with either empty vector (EV) control or SMAD3 overexpression vector (cells were transfected with 1 µg plasmid in 35 mm culture dish). Note that the SMAD3 immunoblot image was acquired at a very low exposure to avoid signal saturation of the overexpression band. b Summarises the outgrowth of SMAD3 overexpression group relative to EV control group of cells in BME matrix. c – d c : SMAD3 and GAPDH (loading control) immunoblots of total cell extracts of control and Pfn1-shRNA expressing MDA-231 cells transiently transfected with the indicated siRNAs. d Summarises the outgrowth of the various transfected groups relative to the control group (control shRNA expressers transfected with control siRNA) in BME matrix. Gene silencing and overexpression-based outgrowth data summarised from two independent experiments, with three technical replicates per experiment). * p

    Techniques Used: Multiple Displacement Amplification, Western Blot, Transfection, Plasmid Preparation, Over Expression, shRNA, Expressing

    Effect of Pfn1 depletion on single cell outgrowth of BCC in 3D BME matrix. a Pfn1 and Pfn2 immunoblot analyses of total cell extracts of MDA-231 cells stably expressing either control or Pfn1-shRNA (GAPDH blot serves as a loading control). b Representative images of outgrowth of control and Pfn1-shRNA MDA-231 cells on 3D BME matrix from an initial seeding of 500 cells/well in a 384-well plate. c A bar graph summarizing the final number of cells (counted on day 10) in control vs Pfn1 shRNA groups for different seeding densities. All data are normalised to the final number of cells in control group for initial seeding density equal to 250 cells/well based on cell-count analyses of 3 technical replicates/condition from two independent experiments. d-g ; d , f Immunoblot analyses of Pfn1 and Pfn2 from total cell extracts of MDA-231 ( d ) and MDA-157 ( f ) cells following transient transfection with the indicated siRNAs (tubulin blot serves as a loading control). The bar graphs in e and g summarise the outgrowth of Pfn1 and Pfn2 KD MDA-231 and MDA-157 cells, respectively, on 3D BME matrix 7 days after seeding relative to the respective control siRNA transfected cell lines (data summarised from two independent experiments with three technical replicates per experiment) * p
    Figure Legend Snippet: Effect of Pfn1 depletion on single cell outgrowth of BCC in 3D BME matrix. a Pfn1 and Pfn2 immunoblot analyses of total cell extracts of MDA-231 cells stably expressing either control or Pfn1-shRNA (GAPDH blot serves as a loading control). b Representative images of outgrowth of control and Pfn1-shRNA MDA-231 cells on 3D BME matrix from an initial seeding of 500 cells/well in a 384-well plate. c A bar graph summarizing the final number of cells (counted on day 10) in control vs Pfn1 shRNA groups for different seeding densities. All data are normalised to the final number of cells in control group for initial seeding density equal to 250 cells/well based on cell-count analyses of 3 technical replicates/condition from two independent experiments. d-g ; d , f Immunoblot analyses of Pfn1 and Pfn2 from total cell extracts of MDA-231 ( d ) and MDA-157 ( f ) cells following transient transfection with the indicated siRNAs (tubulin blot serves as a loading control). The bar graphs in e and g summarise the outgrowth of Pfn1 and Pfn2 KD MDA-231 and MDA-157 cells, respectively, on 3D BME matrix 7 days after seeding relative to the respective control siRNA transfected cell lines (data summarised from two independent experiments with three technical replicates per experiment) * p

    Techniques Used: Multiple Displacement Amplification, Stable Transfection, Expressing, shRNA, Cell Counting, Transfection

    9) Product Images from "Decreased Expression of CoREST1 and CoREST2 Together with LSD1 and HDAC1/2 during Neuronal Differentiation"

    Article Title: Decreased Expression of CoREST1 and CoREST2 Together with LSD1 and HDAC1/2 during Neuronal Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131760

    CoREST1 and CoREST2, but not CoREST3 are down-regulated during NGF-dependent neuronal differentiation of PC12 cells. (A) Left: representative immunoblots of CoREST1, HDAC1 and LSD1 in equivalent protein amounts of cytosolic and nuclei fractions of control (-NGF) and NGF treated (50 mg/ml during 7 days) PC12 cells. Histone H3 was used as loading control for nuclear fraction and GAPDH for cytoplasmic fraction. Right: graph of relative protein levels of CoREST1, HDAC1 and LSD1 respect to histone H3 levels. Values correspond to the mean ± SEM of at least 3 independent experiments. **p
    Figure Legend Snippet: CoREST1 and CoREST2, but not CoREST3 are down-regulated during NGF-dependent neuronal differentiation of PC12 cells. (A) Left: representative immunoblots of CoREST1, HDAC1 and LSD1 in equivalent protein amounts of cytosolic and nuclei fractions of control (-NGF) and NGF treated (50 mg/ml during 7 days) PC12 cells. Histone H3 was used as loading control for nuclear fraction and GAPDH for cytoplasmic fraction. Right: graph of relative protein levels of CoREST1, HDAC1 and LSD1 respect to histone H3 levels. Values correspond to the mean ± SEM of at least 3 independent experiments. **p

    Techniques Used: Western Blot

    10) Product Images from "DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA"

    Article Title: DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1299-x

    DAP5 positively regulates cap-independent translation of DSCR1.4 by binding to 5′UTR of DSCR1.4. a , b DAP5 interacts with hDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated hDSCR1.4 5′UTR constructs were incubated with SHSY5Y cell extracts. The region interacting with DAP5 was confirmed by Western blot. GAPDH and hDSCR1.4 were used as negative control. Nonbiotinylated hDSCR1.4 UTR was used as competitor. c DAP5 binds to proximal 136 nucleotides of mDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated mDSCR1.4 5′UTR transcripts were incubated with N2A cell extracts. Binding region was verified by Western blot. d , e A reduction of DAP5 decreases cap-independent translation efficiency of DSCR1.4 5′UTR. d SHSY5Y and e N2A cells were transfected with control siRNA (siCon) or DAP5 siRNA (siDAP5) and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and siCon transfected cells was set as 1. The bars represent the mean ± SEM ( n = 7, n = 7). f , g DAP5 overexpression upregulated cap-independent translation of DSCR1.4 5′UTR. f SHSY5Y and g N2A cells were transfected with Flag Mock or Flag DAP5 plasmids and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and Flag Mock transfected cells was set as 1. The bars represent the mean ± SEM ( n = 5, n = 5). h , i DAP5 deficiency results in reduction of DSCR1.4 protein levels. siCon or siDAP5 was transfected on h SHSY5Y and i N2A cells. DAP5 knockdown was confirmed by Western blot using anti-DAP5. j , k Overexpressed DAP5 increases DSCR1.4 protein levels. j SHSY5Y and k N2A cells were transfected with Flag Mock or Flag DAP5 and incubated for 24 h. DAP5 overexpression was verified by Western blot using anti-Flag antibody. l The increase of DSCR1.4 expressions by DAP5 overexpression results from cap-independent translation. SHSY5Ycells were transfected with Flag Mock or Flag DAP5 and 18 h later incubated and followed by 200 μM rapamycin treatment for 6 h. The rapamycin activity was analyzed by phosphorylation status of S6RP protein. DAP5 overexpression was confirmed by anti-Flag antibody. Data information: In d – g , * P
    Figure Legend Snippet: DAP5 positively regulates cap-independent translation of DSCR1.4 by binding to 5′UTR of DSCR1.4. a , b DAP5 interacts with hDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated hDSCR1.4 5′UTR constructs were incubated with SHSY5Y cell extracts. The region interacting with DAP5 was confirmed by Western blot. GAPDH and hDSCR1.4 were used as negative control. Nonbiotinylated hDSCR1.4 UTR was used as competitor. c DAP5 binds to proximal 136 nucleotides of mDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated mDSCR1.4 5′UTR transcripts were incubated with N2A cell extracts. Binding region was verified by Western blot. d , e A reduction of DAP5 decreases cap-independent translation efficiency of DSCR1.4 5′UTR. d SHSY5Y and e N2A cells were transfected with control siRNA (siCon) or DAP5 siRNA (siDAP5) and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and siCon transfected cells was set as 1. The bars represent the mean ± SEM ( n = 7, n = 7). f , g DAP5 overexpression upregulated cap-independent translation of DSCR1.4 5′UTR. f SHSY5Y and g N2A cells were transfected with Flag Mock or Flag DAP5 plasmids and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and Flag Mock transfected cells was set as 1. The bars represent the mean ± SEM ( n = 5, n = 5). h , i DAP5 deficiency results in reduction of DSCR1.4 protein levels. siCon or siDAP5 was transfected on h SHSY5Y and i N2A cells. DAP5 knockdown was confirmed by Western blot using anti-DAP5. j , k Overexpressed DAP5 increases DSCR1.4 protein levels. j SHSY5Y and k N2A cells were transfected with Flag Mock or Flag DAP5 and incubated for 24 h. DAP5 overexpression was verified by Western blot using anti-Flag antibody. l The increase of DSCR1.4 expressions by DAP5 overexpression results from cap-independent translation. SHSY5Ycells were transfected with Flag Mock or Flag DAP5 and 18 h later incubated and followed by 200 μM rapamycin treatment for 6 h. The rapamycin activity was analyzed by phosphorylation status of S6RP protein. DAP5 overexpression was confirmed by anti-Flag antibody. Data information: In d – g , * P

    Techniques Used: Binding Assay, In Vitro, Construct, Incubation, Western Blot, Negative Control, Transfection, Luciferase, Activity Assay, Over Expression

    BDNF makes cap-independent translation of DSCR1.4 mRNA more actively by increasing DAP5 expression. a , b BDNF treatment on DIV 3 hippocampal neuron increases protein levels of DAP5 and DSCR1.4 but not DSCR1.4 mRNA level. Vehicle (DDW) or 30 μM BDNF were treated for 1 h. a The protein levels were confirmed by Western blot. GAPDH and phosphorylation of ERK were used as a loading control and marker of BDNF activity, respectively. b Endogenous DSCR1.4 mRNA levels were analyzed by qRT-PCR and were normalized to β-actin. The bars represent the mean ± SEM ( n = 3). c Cap-independent translation is essential for DSCR1.4 protein accumulation by BDNF. DIV 3 hippocampal neurons were treated with vehicle (DMSO), 100 μM RAD001(RAD) or 50 mg/ml cycloheximide (CHX) for 3 h followed by BDNF treatment for 1 h. The levels of each protein were confirmed by Western blot. The numbers at the bottom indicate the fold relative to a vehicle. The amount of DSCR1.4 was normalized to GAPDH. d , e BDNF raises cap-independent translation activity of DSCR1.4 mRNA. At 24 h after d pRF hDSCR1.4 5′UTR or e pRF mDSCR1.4 5′UTR vectors were transfected into DIV 2 hippocampal neurons, Vehicle (DDW) and BDNF were treated to the neurons for 1 h. The bars represent the mean ± SEM ( e ; n = 5, F; n = 3). f BDNF increases the interaction between DAP5 and DSCR1.4 5′UTR. In vitro transcribed biotin-DSCR1.4 5′UTR was incubated with extracts of the vehicle (DDW) or 30 μM BDNF-treated DIV 3 mouse hippocampal neurons. DAP5 binding was measured by Western blot. Phospho-ERK was used to confirm the activity of BDNF. GADPH was used as a loading control and negative control. g , h BDNF increases the cap-independent local translation of DSCR1.4 mRNA in axon as well as soma. EGFP and pRF mDSCR1.4 5′ 3′ UTR vectors were co-transfected into DIV 2 mouse hippocampal neurons. At 24 h later, 100 μM anisomycin was treated for 3 h and then 30 μM BDNF was treated for 1 h, followed by 5 μM puromycin treatment for 40 min. To detect newly synthesized FLUC proteins, Puro-PLA assay was conducted. g Representative image obtained from confocal microscopy. h The graph shows relative fluorescence intensity measured by Image J. The bars represent the mean ± SEM (Vehicle; n = 11, Anisomycin; n = 12, BDNF; n = 11, BDNF + Anisomycin; n = 11). Scale bar, 30 μm. Data information: In d , e , h , * P
    Figure Legend Snippet: BDNF makes cap-independent translation of DSCR1.4 mRNA more actively by increasing DAP5 expression. a , b BDNF treatment on DIV 3 hippocampal neuron increases protein levels of DAP5 and DSCR1.4 but not DSCR1.4 mRNA level. Vehicle (DDW) or 30 μM BDNF were treated for 1 h. a The protein levels were confirmed by Western blot. GAPDH and phosphorylation of ERK were used as a loading control and marker of BDNF activity, respectively. b Endogenous DSCR1.4 mRNA levels were analyzed by qRT-PCR and were normalized to β-actin. The bars represent the mean ± SEM ( n = 3). c Cap-independent translation is essential for DSCR1.4 protein accumulation by BDNF. DIV 3 hippocampal neurons were treated with vehicle (DMSO), 100 μM RAD001(RAD) or 50 mg/ml cycloheximide (CHX) for 3 h followed by BDNF treatment for 1 h. The levels of each protein were confirmed by Western blot. The numbers at the bottom indicate the fold relative to a vehicle. The amount of DSCR1.4 was normalized to GAPDH. d , e BDNF raises cap-independent translation activity of DSCR1.4 mRNA. At 24 h after d pRF hDSCR1.4 5′UTR or e pRF mDSCR1.4 5′UTR vectors were transfected into DIV 2 hippocampal neurons, Vehicle (DDW) and BDNF were treated to the neurons for 1 h. The bars represent the mean ± SEM ( e ; n = 5, F; n = 3). f BDNF increases the interaction between DAP5 and DSCR1.4 5′UTR. In vitro transcribed biotin-DSCR1.4 5′UTR was incubated with extracts of the vehicle (DDW) or 30 μM BDNF-treated DIV 3 mouse hippocampal neurons. DAP5 binding was measured by Western blot. Phospho-ERK was used to confirm the activity of BDNF. GADPH was used as a loading control and negative control. g , h BDNF increases the cap-independent local translation of DSCR1.4 mRNA in axon as well as soma. EGFP and pRF mDSCR1.4 5′ 3′ UTR vectors were co-transfected into DIV 2 mouse hippocampal neurons. At 24 h later, 100 μM anisomycin was treated for 3 h and then 30 μM BDNF was treated for 1 h, followed by 5 μM puromycin treatment for 40 min. To detect newly synthesized FLUC proteins, Puro-PLA assay was conducted. g Representative image obtained from confocal microscopy. h The graph shows relative fluorescence intensity measured by Image J. The bars represent the mean ± SEM (Vehicle; n = 11, Anisomycin; n = 12, BDNF; n = 11, BDNF + Anisomycin; n = 11). Scale bar, 30 μm. Data information: In d , e , h , * P

    Techniques Used: Expressing, Western Blot, Marker, Activity Assay, Quantitative RT-PCR, Transfection, In Vitro, Incubation, Binding Assay, Negative Control, Synthesized, Proximity Ligation Assay, Confocal Microscopy, Fluorescence

    hDSCR1.4 and mDSCR1.4 mRNA are cap-independently translated and have cis-regulatory elements in their 5′UTRs. a Cap-independent translational regulation contributes to DSCR1.4 protein expression. SHSY5Y cells were treated with DMSO or 200 μM rapamycin or 50 mg/ml cycloheximide for the indicated times. The levels of endogenous proteins were measured by Western blotting (WB) using anti-DSCR1.4, anti-phosphoS6RP, anti-GAPDH antibodies. GAPDH was used as a loading control. The activity of rapamycin was analyzed by the phosphorylation status of S6RP. The numbers at the bottom indicate the fold increases relative to control. The amount of DSCR1.4 was normalized to GAPDH. b Schematic representation of pRF bicistronic luciferase plasmids used for observing cap-independent translation activity of human and mouse DSCR1.4 5′UTR. c , d hDSCR1.4 5′UTR and mDSCR1.4 5′UTR induce cap-independent translation initiation. c SHSY5Y and d N2A cells were transfected with the bicistronic reporter plasmids and were incubated for 24 h. pRF β-globin was used as a negative control. pRF EMCV and pRF p53 were used as the positive control. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 4). e , f hDSCR1.4 5′UTR and mDSCR1.4 5′UTR prefer cap-independent translation to cap-dependent translation. e SHSY5Y cells were transfected with in vitro transcribed m7G capped or ApppG capped hDSCR1.4 5′UTR-FLUC. f N2A cells were transfected with in vitro transcribed m7G capped or ApppG capped mDSCR1.4 5′UTR-FLUC. Transfected cells were incubated for 6 h and were harvested. Translation activity is shown as the ratio of FLUC to FLUC mRNA. Translation activity of m7G capped transcripts was set as 1. The bars represent the mean ± SEM ( n = 4, n = 3). g , i The 5′ proximal 119 nucleotides sequence of hDSCR1.4 5′UTR is important for cap-independent translation activity of hDSCR1.4 5′UTR. pRF plasmids with serial deletion constructs and six nucleotides mutant construct were transfected into g SHSY5Y cells and i mouse primary hippocampal neurons. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3). h , j The 5′ proximal 136 nucleotides are essential for cap-independent translation activity of mDSCR1.4 5′UTR. Indicated pRF plasmids were transfected into h N2A cells and j mouse hippocampal neurons. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 3). Data information: In c – j , * P
    Figure Legend Snippet: hDSCR1.4 and mDSCR1.4 mRNA are cap-independently translated and have cis-regulatory elements in their 5′UTRs. a Cap-independent translational regulation contributes to DSCR1.4 protein expression. SHSY5Y cells were treated with DMSO or 200 μM rapamycin or 50 mg/ml cycloheximide for the indicated times. The levels of endogenous proteins were measured by Western blotting (WB) using anti-DSCR1.4, anti-phosphoS6RP, anti-GAPDH antibodies. GAPDH was used as a loading control. The activity of rapamycin was analyzed by the phosphorylation status of S6RP. The numbers at the bottom indicate the fold increases relative to control. The amount of DSCR1.4 was normalized to GAPDH. b Schematic representation of pRF bicistronic luciferase plasmids used for observing cap-independent translation activity of human and mouse DSCR1.4 5′UTR. c , d hDSCR1.4 5′UTR and mDSCR1.4 5′UTR induce cap-independent translation initiation. c SHSY5Y and d N2A cells were transfected with the bicistronic reporter plasmids and were incubated for 24 h. pRF β-globin was used as a negative control. pRF EMCV and pRF p53 were used as the positive control. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 4). e , f hDSCR1.4 5′UTR and mDSCR1.4 5′UTR prefer cap-independent translation to cap-dependent translation. e SHSY5Y cells were transfected with in vitro transcribed m7G capped or ApppG capped hDSCR1.4 5′UTR-FLUC. f N2A cells were transfected with in vitro transcribed m7G capped or ApppG capped mDSCR1.4 5′UTR-FLUC. Transfected cells were incubated for 6 h and were harvested. Translation activity is shown as the ratio of FLUC to FLUC mRNA. Translation activity of m7G capped transcripts was set as 1. The bars represent the mean ± SEM ( n = 4, n = 3). g , i The 5′ proximal 119 nucleotides sequence of hDSCR1.4 5′UTR is important for cap-independent translation activity of hDSCR1.4 5′UTR. pRF plasmids with serial deletion constructs and six nucleotides mutant construct were transfected into g SHSY5Y cells and i mouse primary hippocampal neurons. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3). h , j The 5′ proximal 136 nucleotides are essential for cap-independent translation activity of mDSCR1.4 5′UTR. Indicated pRF plasmids were transfected into h N2A cells and j mouse hippocampal neurons. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 3). Data information: In c – j , * P

    Techniques Used: Expressing, Western Blot, Activity Assay, Luciferase, Transfection, Incubation, Negative Control, Positive Control, Plasmid Preparation, In Vitro, Sequencing, Construct, Mutagenesis

    11) Product Images from "A Critical Role of Autophagy in Regulating Microglia Polarization in Neurodegeneration"

    Article Title: A Critical Role of Autophagy in Regulating Microglia Polarization in Neurodegeneration

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2018.00378

    TNF-α disrupted the autophagic flux in microglia. (A) Dose-dependent effect of the lysosome inhibitor BafA1 treatment for 2 h on LC3II accumulation in BV2 cells, as evaluated by western blotting. N = 3. (B) Effect of TNF-α on LC3II levels during lysosome inhibition. BV2 cells were treated with 5 ng/ml TNF-α for 24 h, and then added with BafA1(50 nM) for 2 h before subjected to western blotting. Actin served as loading controls in panels A,B . N = 4. (C,D) Autophagic assay in microglia treated with TNF-α, BafA1, or in combination. BV2 cells were transfected with RFP-GFP-tandem fluorescent LC3 cDNA for 24 h before treatment. Confocal microscope pictures showing yellow (GFP and RFP overlap) and red LC3 puncta formation in different groups. Scale bar, 10 μm. LC3 dots were visualized and quantified from at least 30 cells per group. (E) Effect of TNF-α on lysosomal biogenesis. BV2 cells were treated with 5 ng/ml TNF-α for 3, 12, or 24 h. The cytosolic and nuclear fractions were subjected to western blotting analysis of TFEB, with GAPDH and Histone 2B as the cytosolic and nuclear loading controls, respectively. (F,G) Effect of TNF-α on lysosomal protein LAMP1 (F) and LAMP2 (G) levels in BV2 cells. N = 3. ∗ P
    Figure Legend Snippet: TNF-α disrupted the autophagic flux in microglia. (A) Dose-dependent effect of the lysosome inhibitor BafA1 treatment for 2 h on LC3II accumulation in BV2 cells, as evaluated by western blotting. N = 3. (B) Effect of TNF-α on LC3II levels during lysosome inhibition. BV2 cells were treated with 5 ng/ml TNF-α for 24 h, and then added with BafA1(50 nM) for 2 h before subjected to western blotting. Actin served as loading controls in panels A,B . N = 4. (C,D) Autophagic assay in microglia treated with TNF-α, BafA1, or in combination. BV2 cells were transfected with RFP-GFP-tandem fluorescent LC3 cDNA for 24 h before treatment. Confocal microscope pictures showing yellow (GFP and RFP overlap) and red LC3 puncta formation in different groups. Scale bar, 10 μm. LC3 dots were visualized and quantified from at least 30 cells per group. (E) Effect of TNF-α on lysosomal biogenesis. BV2 cells were treated with 5 ng/ml TNF-α for 3, 12, or 24 h. The cytosolic and nuclear fractions were subjected to western blotting analysis of TFEB, with GAPDH and Histone 2B as the cytosolic and nuclear loading controls, respectively. (F,G) Effect of TNF-α on lysosomal protein LAMP1 (F) and LAMP2 (G) levels in BV2 cells. N = 3. ∗ P

    Techniques Used: Western Blot, Inhibition, Transfection, Microscopy

    12) Product Images from "MicroRNA-93 Regulates Hypoxia-Induced Autophagy by Targeting ULK1"

    Article Title: MicroRNA-93 Regulates Hypoxia-Induced Autophagy by Targeting ULK1

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/2709053

    The knockdown of ULK1 by siRNA suppressed hypoxia-induced autophagy. (a, b) MEFs and CHO cells were transfected with three pairs of siRNAs to ULK for 24 h, respectively, si-ULK1 (488), si-ULK1 (2457), and si-ULK1 (3310), setting NC as a control. Cell lysates were prepared and subjected to Western blot analysis by using anti-ULK1 and anti-Gapdh. The densitometric ratios from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (c) Si-ULK1 (488) was transfected into MEFs for 12 h, followed by hypoxic treatment or normoxic treatment for another 24 h. Cell lysates were analyzed by Western blot using anti-ULK1, anti-P62, anti-LC3, and anti-Gapdh. Densitometric ratios of the samples were quantified by using ImageJ. (d) MEFs were treated as the same as (c). Cells were fixed and immunostained by anti-ULK1 (green) and anti-P62 (red) antibodies. Scale bar, 20 μ m.
    Figure Legend Snippet: The knockdown of ULK1 by siRNA suppressed hypoxia-induced autophagy. (a, b) MEFs and CHO cells were transfected with three pairs of siRNAs to ULK for 24 h, respectively, si-ULK1 (488), si-ULK1 (2457), and si-ULK1 (3310), setting NC as a control. Cell lysates were prepared and subjected to Western blot analysis by using anti-ULK1 and anti-Gapdh. The densitometric ratios from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (c) Si-ULK1 (488) was transfected into MEFs for 12 h, followed by hypoxic treatment or normoxic treatment for another 24 h. Cell lysates were analyzed by Western blot using anti-ULK1, anti-P62, anti-LC3, and anti-Gapdh. Densitometric ratios of the samples were quantified by using ImageJ. (d) MEFs were treated as the same as (c). Cells were fixed and immunostained by anti-ULK1 (green) and anti-P62 (red) antibodies. Scale bar, 20 μ m.

    Techniques Used: Transfection, Western Blot

    13) Product Images from "Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers"

    Article Title: Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers

    Journal: Autophagy

    doi: 10.4161/15548627.2014.981788

    For figure legend, see page 2133. Figure 5 ( See previous page ). The transcription co-activator YAP1 is a key player in the autophagy-dependent proliferation and invasion of TN BC cells. ( A ) Western blots showing phosphorylated-YAP1 (P-YAP1), YAP1, and AP2A1 proteins in stable cell lines following 3D culture. AP2A1 is used as internal control for protein loading. The bar graph (right panel) shows the corresponding quantification of P-YAP1/YAP1 protein level ratios. ( B ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3D culture. GAPDH is used as an internal control for total mRNA expression. ( C ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with 3-methyladenine (3-MA; 20 mM) for 3 h. ( D ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3-MA treatment. GAPDH is used as an internal control for total mRNA expression. ( E ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with rapamycin (2 μM) for 3 h. ( F ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following rapamycin treatment. GAPDH is used as an internal control for total mRNA expression. ( G ) Left, western blots showing YAP1 protein levels in MDA231 cells after transfection with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. AP2A1 is used as an internal control for protein loading. Middle, representative bright field images from Control- or YAP1-depleted cells cultured in 3D, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Right, the bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( H ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from MDA231 cells transiently transfected with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. for 72 h prior to the assay. Left histogram: Numbers of invading cells, which passed through a Transwell over 6 h of incubation. Right panel: Percentage of cells, relative to siCtrl (100%), which passed through a Transwell over 6 h of incubation. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( I ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from autophagy-deficient MDA231 cells (36 h of transfection with si ATG7 or si ATG5 , as indicated), transfected again (36 h) with an empty vector or a vector expressing YAP1-S127A, a nonphosphorylable mutant form of YAP1. Numbers of invading cells, which passed through a Transwell over 6 h of incubation, are shown. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( J ) Results are from autophagy-proficient (sh Ctrl ) or autophagy-deficient (sh ATG5 ) MDA231 cells, transfected with an empty vector or a vector expressing YAP1-S127A, after 3 d of 3D culture. Representative bright field images from ATG5-depleted cells expressing or not YAP1-S127A, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 5 independent experiments). P -values are based on the Student t test.
    Figure Legend Snippet: For figure legend, see page 2133. Figure 5 ( See previous page ). The transcription co-activator YAP1 is a key player in the autophagy-dependent proliferation and invasion of TN BC cells. ( A ) Western blots showing phosphorylated-YAP1 (P-YAP1), YAP1, and AP2A1 proteins in stable cell lines following 3D culture. AP2A1 is used as internal control for protein loading. The bar graph (right panel) shows the corresponding quantification of P-YAP1/YAP1 protein level ratios. ( B ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3D culture. GAPDH is used as an internal control for total mRNA expression. ( C ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with 3-methyladenine (3-MA; 20 mM) for 3 h. ( D ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following 3-MA treatment. GAPDH is used as an internal control for total mRNA expression. ( E ) Western blots (left) and corresponding quantification (bar graph, right) showing phosphorylated-YAP1 (P-YAP1), YAP1 and AP2A1 proteins in MDA231 cells cultured during 3 d in 3D and then either untreated (-) or treated with rapamycin (2 μM) for 3 h. ( F ) mRNA levels of YAP1-target genes were monitored by RT-qPCR following rapamycin treatment. GAPDH is used as an internal control for total mRNA expression. ( G ) Left, western blots showing YAP1 protein levels in MDA231 cells after transfection with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. AP2A1 is used as an internal control for protein loading. Middle, representative bright field images from Control- or YAP1-depleted cells cultured in 3D, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Right, the bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( H ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from MDA231 cells transiently transfected with control- (si Ctrl ) or YAP1-targeted (si YAP1 ) siRNA. for 72 h prior to the assay. Left histogram: Numbers of invading cells, which passed through a Transwell over 6 h of incubation. Right panel: Percentage of cells, relative to siCtrl (100%), which passed through a Transwell over 6 h of incubation. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( I ) Invasion assay using a BioCoat TM Matrigel TM Invasion Chamber from autophagy-deficient MDA231 cells (36 h of transfection with si ATG7 or si ATG5 , as indicated), transfected again (36 h) with an empty vector or a vector expressing YAP1-S127A, a nonphosphorylable mutant form of YAP1. Numbers of invading cells, which passed through a Transwell over 6 h of incubation, are shown. Data are shown as means +/− sem (N = 3 independent experiments). p-values are based on the Student t test. ( J ) Results are from autophagy-proficient (sh Ctrl ) or autophagy-deficient (sh ATG5 ) MDA231 cells, transfected with an empty vector or a vector expressing YAP1-S127A, after 3 d of 3D culture. Representative bright field images from ATG5-depleted cells expressing or not YAP1-S127A, as indicated. Scale bars = 100 μm (low magnification) and 50 μm (high magnification). Bar graphs represent the total area covered by the stellate structures per field (left panel) and the area per clone (right panel). Data are shown as means +/− sem (N = 5 independent experiments). P -values are based on the Student t test.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, Stable Transfection, Quantitative RT-PCR, Expressing, Cell Culture, Transfection, Invasion Assay, Incubation, Plasmid Preparation, Mutagenesis

    14) Product Images from "GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3"

    Article Title: GATA4 promotes hepatoblastoma cell proliferation by altering expression of miR125b and DKK3

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12839

    GATA4 trans-inactivates miR125b expression and indirectly regulates DKK3 expression A. Schematic illustration of 5 putative wild-type miR125b promoter constructs fused to a luciferase reporter gene using pGL-3 plasmid vector. B. Luciferase activity in extracts in Huh6 cells treated with or without GATA4 siRNA (Huh6 GATA4 siRNA) and transiently transfected with the 5 wild-type miR125b luciferase reporter constructs. Luciferase values are normalized to the Huh6 cells transfected with the empty pGL-3 basic vector. C. Schematic illustration of human wild-type or mutant miR125b-3 promotor constructs fused to a luciferase reporter gene. D. Luciferase activity in extracts from Huh6 cells treated with or without GATA4 shRNA cells and transiently transfected with the wild-type or mutant miR125b-3 luciferase reporter constructs. Average activity from five repeated samples was used to calculate luciferase activity. E. Schematic of the miR125b-3 upstream promoter containing a single GATA4-binding site. ChIP analyses revealed that GATA4 binds to the upstream GATA boxes in Huh6 cells; binding activity decreased in Huh6 cells treated with GATA4 siRNA. F. Western blot analysis of DKK3 and GATA4 expression in Huh6 cells treated with or without GATA4 siRNA cells; GAPDH served as the control. Huh6 NC: Huh6 cells treated with negative control miRNA. * P
    Figure Legend Snippet: GATA4 trans-inactivates miR125b expression and indirectly regulates DKK3 expression A. Schematic illustration of 5 putative wild-type miR125b promoter constructs fused to a luciferase reporter gene using pGL-3 plasmid vector. B. Luciferase activity in extracts in Huh6 cells treated with or without GATA4 siRNA (Huh6 GATA4 siRNA) and transiently transfected with the 5 wild-type miR125b luciferase reporter constructs. Luciferase values are normalized to the Huh6 cells transfected with the empty pGL-3 basic vector. C. Schematic illustration of human wild-type or mutant miR125b-3 promotor constructs fused to a luciferase reporter gene. D. Luciferase activity in extracts from Huh6 cells treated with or without GATA4 shRNA cells and transiently transfected with the wild-type or mutant miR125b-3 luciferase reporter constructs. Average activity from five repeated samples was used to calculate luciferase activity. E. Schematic of the miR125b-3 upstream promoter containing a single GATA4-binding site. ChIP analyses revealed that GATA4 binds to the upstream GATA boxes in Huh6 cells; binding activity decreased in Huh6 cells treated with GATA4 siRNA. F. Western blot analysis of DKK3 and GATA4 expression in Huh6 cells treated with or without GATA4 siRNA cells; GAPDH served as the control. Huh6 NC: Huh6 cells treated with negative control miRNA. * P

    Techniques Used: Expressing, Construct, Luciferase, Plasmid Preparation, Activity Assay, Transfection, Mutagenesis, shRNA, Binding Assay, Chromatin Immunoprecipitation, Western Blot, Negative Control

    15) Product Images from "Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation"

    Article Title: Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-06-0401

    Graded control over the expression of a constitutively active form of MLCK (CA-MLCK) and ROCK2 (CA-ROCK2). (A) Schematic of doxycycline-inducible lentiviral system, where X encodes MLCK, ROCK1, or ROCK2. (B) Representative Western blot showing expression levels of endogenous MLCK, CA-MLCK, and GAPDH in U2OS (left) and U373MG cells (right) as a function of doxycycline concentration. U2OS cells were probed with rabbit ant-MLCK (Abcam 76092) and U373MG cells were probed with mouse anti-MLCK (Sigma M7905). Expression levels of CA-MLCK were quantified, normalized to GAPDH and to the highest doxycycline concentration for each cell line, and plotted below the respective Western blots ( n = 4 blots for U2OS and n = 6 blots for U373MG). (C) Representative Western blot showing expression levels of endogenous ROCK2, CA-ROCK2, and GAPDH in U2OS (left) and U373MG (right) cells in the presence of various amounts of doxycycline. Expression levels of CA-ROCK2 were quantified, normalized to GAPDH and to the highest doxycycline concentration, and plotted below the respective Western blots ( n = 10 blots for U2OS and n = 10 blots for U373MG at the maximum doxycycline concentration). Statistical parameters shown represent the Spearman’s rank correlation coefficient (ρ) and p value.
    Figure Legend Snippet: Graded control over the expression of a constitutively active form of MLCK (CA-MLCK) and ROCK2 (CA-ROCK2). (A) Schematic of doxycycline-inducible lentiviral system, where X encodes MLCK, ROCK1, or ROCK2. (B) Representative Western blot showing expression levels of endogenous MLCK, CA-MLCK, and GAPDH in U2OS (left) and U373MG cells (right) as a function of doxycycline concentration. U2OS cells were probed with rabbit ant-MLCK (Abcam 76092) and U373MG cells were probed with mouse anti-MLCK (Sigma M7905). Expression levels of CA-MLCK were quantified, normalized to GAPDH and to the highest doxycycline concentration for each cell line, and plotted below the respective Western blots ( n = 4 blots for U2OS and n = 6 blots for U373MG). (C) Representative Western blot showing expression levels of endogenous ROCK2, CA-ROCK2, and GAPDH in U2OS (left) and U373MG (right) cells in the presence of various amounts of doxycycline. Expression levels of CA-ROCK2 were quantified, normalized to GAPDH and to the highest doxycycline concentration, and plotted below the respective Western blots ( n = 10 blots for U2OS and n = 10 blots for U373MG at the maximum doxycycline concentration). Statistical parameters shown represent the Spearman’s rank correlation coefficient (ρ) and p value.

    Techniques Used: Expressing, Western Blot, Concentration Assay

    Graded increases in CA-MLCK and CA-ROCK2 produce graded changes in p-RLC and pp-RLC. (A) Representative Western blots probed for p-RLC and pp-RLC in U2OS CA-MLCK (top) and U373MG CA-MLCK (bottom). Phosphorylation levels were quantified, normalized to GAPDH and CA-MLCK + 0 ng/ml doxycycline for each cell line, and plotted below the respective Western blots. p-RLC is shown by empty gray circles, whereas pp-RLC is shown by black triangles (U2OS: n = 6 blots for p-RLC [mouse] and 7 blots for pp-RLC [rabbit] blots; U373MG: n = 8 blots for p-RLC [mouse] and n = 9 blots for pp-RLC [rabbit]). (B) Representative Western blots probed for pp-RLC and p-MCL in U2OS CA-ROCK2 (top) and U373MG CA-ROCK2 (bottom). Phosphorylation levels were quantified, normalized to GAPDH and CA-ROCK2 + 0 ng/ml doxycycline for each cell line, and plotted below the respective Western blots. p-RLC is shown as empty, black circles, whereas pp-RLC is shown as solid, black triangles (U2OS: n = 4 blots for p-RLC [mouse] and n = 11 blots for pp-RLC [rabbit] expression; U373MG: n = 9 blots for pp-RLC [rabbit] and n = 6 blots for p-RLC [mouse]). Statistical parameters shown represent the Spearman’s rank correlation coefficient (ρ) and p value.
    Figure Legend Snippet: Graded increases in CA-MLCK and CA-ROCK2 produce graded changes in p-RLC and pp-RLC. (A) Representative Western blots probed for p-RLC and pp-RLC in U2OS CA-MLCK (top) and U373MG CA-MLCK (bottom). Phosphorylation levels were quantified, normalized to GAPDH and CA-MLCK + 0 ng/ml doxycycline for each cell line, and plotted below the respective Western blots. p-RLC is shown by empty gray circles, whereas pp-RLC is shown by black triangles (U2OS: n = 6 blots for p-RLC [mouse] and 7 blots for pp-RLC [rabbit] blots; U373MG: n = 8 blots for p-RLC [mouse] and n = 9 blots for pp-RLC [rabbit]). (B) Representative Western blots probed for pp-RLC and p-MCL in U2OS CA-ROCK2 (top) and U373MG CA-ROCK2 (bottom). Phosphorylation levels were quantified, normalized to GAPDH and CA-ROCK2 + 0 ng/ml doxycycline for each cell line, and plotted below the respective Western blots. p-RLC is shown as empty, black circles, whereas pp-RLC is shown as solid, black triangles (U2OS: n = 4 blots for p-RLC [mouse] and n = 11 blots for pp-RLC [rabbit] expression; U373MG: n = 9 blots for pp-RLC [rabbit] and n = 6 blots for p-RLC [mouse]). Statistical parameters shown represent the Spearman’s rank correlation coefficient (ρ) and p value.

    Techniques Used: Western Blot, Expressing

    16) Product Images from ""

    Article Title:

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA118.000946

    Immunoblot validation of the abundance of differentially accumulating epididymosome proteins. A ,, Quantitative MS data were validated via immunoblotting of differentially accumulating proteins. Candidate proteins included representatives with the highest abundance (according to TMT reporter ion intensity) in epididymosomes from the proximal segment of the epididymis (caput) (ADAM7, B4GALT1, HSP90B1, MFGE8, PDIA6) in addition to proteins exhibiting increasing accumulation in cauda epididymosomes ( i.e. , ALDH2, CLU, PROM2, BAG6), and those that remained at relatively constant levels in epididymosomes sampled throughout the epididymis (PSMD7, DNM2, HSPA2). B ,, Corresponding MS quantification data are presented. C ,, Negative controls included sperm proteins of testicular origin (IZUMO1, ADAM3, ODF2), D ,, whereas positive controls included validated epididymosome/exosome proteins (GAPDH, FLOT1). Analyses were performed in triplicate using biological samples comprising pooled epididymosomes purified from 12 mice and representative immunoblots are depicted. E ,, A linear regression was performed to compare the quantification data obtained via TMT ( x , axis) and immunoblotting ( y , axis) analyses for each of the targeted epididymosome proteins, revealing significant correlation ( R , 2 = 0.61; p ,
    Figure Legend Snippet: Immunoblot validation of the abundance of differentially accumulating epididymosome proteins. A ,, Quantitative MS data were validated via immunoblotting of differentially accumulating proteins. Candidate proteins included representatives with the highest abundance (according to TMT reporter ion intensity) in epididymosomes from the proximal segment of the epididymis (caput) (ADAM7, B4GALT1, HSP90B1, MFGE8, PDIA6) in addition to proteins exhibiting increasing accumulation in cauda epididymosomes ( i.e. , ALDH2, CLU, PROM2, BAG6), and those that remained at relatively constant levels in epididymosomes sampled throughout the epididymis (PSMD7, DNM2, HSPA2). B ,, Corresponding MS quantification data are presented. C ,, Negative controls included sperm proteins of testicular origin (IZUMO1, ADAM3, ODF2), D ,, whereas positive controls included validated epididymosome/exosome proteins (GAPDH, FLOT1). Analyses were performed in triplicate using biological samples comprising pooled epididymosomes purified from 12 mice and representative immunoblots are depicted. E ,, A linear regression was performed to compare the quantification data obtained via TMT ( x , axis) and immunoblotting ( y , axis) analyses for each of the targeted epididymosome proteins, revealing significant correlation ( R , 2 = 0.61; p ,

    Techniques Used: Mass Spectrometry, Purification, Mouse Assay, Western Blot

    17) Product Images from "Long QT syndrome caveolin‐3 mutations differentially modulate Kv4 and Cav1.2 channels to contribute to action potential prolongation"

    Article Title: Long QT syndrome caveolin‐3 mutations differentially modulate Kv4 and Cav1.2 channels to contribute to action potential prolongation

    Journal: The Journal of Physiology

    doi: 10.1113/JP276014

    Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.
    Figure Legend Snippet: Expression of Cav3‐WT, ‐F97C or ‐S141R with Ca v 1.2 channels in HEK293 cells HEK293 cells were transfected with Cav3‐WT, Cav3‐F97C or Cav3‐S141R with Ca v 1.2 + Ca v β 2cN4 subunits. Cells were immunolabelled with anti‐Cav‐3 (red) and anti‐HA (green) antibodies and imaged using confocal microscopy ( A – I ). J , representative western blot from HEK293 cells expressing Ca V 1.2 and Ca v β 2CN4 subunits with Cav3‐WT, F97C or S141R plasmids probed for Cav3 protein expression with GAPDH loading control. Lysates from HEK293 cells with no plasmid transfected served as a negative control. K , normalized Cav3 protein signals (Cav3/GAPDH) were analysed using ANOVA and were not statistically different between transfected groups.

    Techniques Used: Expressing, Transfection, Confocal Microscopy, Western Blot, Plasmid Preparation, Negative Control

    18) Product Images from "Nek1 and TAZ Interact to Maintain Normal Levels of Polycystin 2"

    Article Title: Nek1 and TAZ Interact to Maintain Normal Levels of Polycystin 2

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2010090992

    Nek1 and TAZ form a negative feedback loop in the regulation of PC2 levels. (A) 293T cells were transfected with Myc-tagged Nek1 along with GST or GST-TAZ. Western blot was performed for GST, Myc, and GAPDH. (B) Immunoprecipitation was performed with
    Figure Legend Snippet: Nek1 and TAZ form a negative feedback loop in the regulation of PC2 levels. (A) 293T cells were transfected with Myc-tagged Nek1 along with GST or GST-TAZ. Western blot was performed for GST, Myc, and GAPDH. (B) Immunoprecipitation was performed with

    Techniques Used: Transfection, Western Blot, Immunoprecipitation

    19) Product Images from "Helicobacter pylori-induced Sonic Hedgehog expression is regulated by NFκB pathway activation: The use of a novel in vitro model to study epithelial response to infection"

    Article Title: Helicobacter pylori-induced Sonic Hedgehog expression is regulated by NFκB pathway activation: The use of a novel in vitro model to study epithelial response to infection

    Journal: Helicobacter

    doi: 10.1111/hel.12152

    H. pylori -induced NFκB activation and Shh expression (A) NFκB status was measured using protein lysates extracted from organoids infected with H. pylori for 0, 2, 4, and 24 hours. Changes in IκBα, IKKα and GAPDH expression were measured by western blot. Quantification of Changes in IκBα relative to GAPDH is shown in B . Data are expressed as the mean + SEM, where *P
    Figure Legend Snippet: H. pylori -induced NFκB activation and Shh expression (A) NFκB status was measured using protein lysates extracted from organoids infected with H. pylori for 0, 2, 4, and 24 hours. Changes in IκBα, IKKα and GAPDH expression were measured by western blot. Quantification of Changes in IκBα relative to GAPDH is shown in B . Data are expressed as the mean + SEM, where *P

    Techniques Used: Activation Assay, Expressing, Infection, Western Blot

    20) Product Images from "Preclinical efficacy for a novel tyrosine kinase inhibitor, ArQule 531 against acute myeloid leukemia"

    Article Title: Preclinical efficacy for a novel tyrosine kinase inhibitor, ArQule 531 against acute myeloid leukemia

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-019-0821-7

    ARQ 531 modulates AML pro-survival kinases. Immunoblot analysis for MOLM-13, MV4-11, and OCI-AML3 AML cell lines (representative of 2-3 independent blots). All cell lines were serum starved overnight followed by 10-min treatment of five to 10 million cells with DMSO, increasing concentrations of ARQ 531 in comparison with 50 nM of quizartinib (Quiz.) or 50 nM gilteritinib (Gilt.), 0.1 μM midostaurin (Mido.), 0.5 μM dasatinib (Das.), 1 μM entospletinib (Ento.), or a combination of 1 μM ARQ 531 and Ento. Twenty to 25 μg of total protein lysate was loaded per lane. GAPDH, HSP90, or β-Actin were used as loading controls. a, b Modulation of phosphorylated SFK and downstream targets in FLT3-ITD cell lines MOLM-13 and MV-11, respectively. c , d Modulation of phosphorylated SYK and BTK in in all three cell lines
    Figure Legend Snippet: ARQ 531 modulates AML pro-survival kinases. Immunoblot analysis for MOLM-13, MV4-11, and OCI-AML3 AML cell lines (representative of 2-3 independent blots). All cell lines were serum starved overnight followed by 10-min treatment of five to 10 million cells with DMSO, increasing concentrations of ARQ 531 in comparison with 50 nM of quizartinib (Quiz.) or 50 nM gilteritinib (Gilt.), 0.1 μM midostaurin (Mido.), 0.5 μM dasatinib (Das.), 1 μM entospletinib (Ento.), or a combination of 1 μM ARQ 531 and Ento. Twenty to 25 μg of total protein lysate was loaded per lane. GAPDH, HSP90, or β-Actin were used as loading controls. a, b Modulation of phosphorylated SFK and downstream targets in FLT3-ITD cell lines MOLM-13 and MV-11, respectively. c , d Modulation of phosphorylated SYK and BTK in in all three cell lines

    Techniques Used:

    21) Product Images from "The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma"

    Article Title: The transcription elongation factor TCEA3 induces apoptosis in rhabdomyosarcoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2258-x

    Characterization of apoptosis triggered by TCEA3 expression. a RH30 cells expressing TCEA3 or empty vector control were harvested for RNA. BAX and BCL2 gene expression were assayed by qRT-PCR. Scale bars 50 µm. b RH30 cells expressing TCEA3 or empty vector were assayed by western blot with antibodies against the indicated apoptotic marker proteins. GAPDH was used as a loading control. c RH30 cells expressing TCEA3 and empty vector were treated with caspase 8 inhibitor (Z-IETD-FMK, 40 μM), caspase 9 inhibitor (Z-LEHD-FMK, 40 μM) or a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 18 h. Immunofluorescence assay was done with anti TCEA3 antibody (green) and anti-cleaved caspase 3 antibody (red). DAPI (blue) was used to visualize nuclei. Scale bar is 50 µm. d Western blot assay on the same cells as in C. to confirm the protein expression of TCEA3, caspase 3 and tubulin as a loading control. * marks cleaved caspase 3. Error bars are S.E.M. Student t test; *** p
    Figure Legend Snippet: Characterization of apoptosis triggered by TCEA3 expression. a RH30 cells expressing TCEA3 or empty vector control were harvested for RNA. BAX and BCL2 gene expression were assayed by qRT-PCR. Scale bars 50 µm. b RH30 cells expressing TCEA3 or empty vector were assayed by western blot with antibodies against the indicated apoptotic marker proteins. GAPDH was used as a loading control. c RH30 cells expressing TCEA3 and empty vector were treated with caspase 8 inhibitor (Z-IETD-FMK, 40 μM), caspase 9 inhibitor (Z-LEHD-FMK, 40 μM) or a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 18 h. Immunofluorescence assay was done with anti TCEA3 antibody (green) and anti-cleaved caspase 3 antibody (red). DAPI (blue) was used to visualize nuclei. Scale bar is 50 µm. d Western blot assay on the same cells as in C. to confirm the protein expression of TCEA3, caspase 3 and tubulin as a loading control. * marks cleaved caspase 3. Error bars are S.E.M. Student t test; *** p

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Marker, Immunofluorescence

    22) Product Images from "A three-way inter-molecular network accounts for the CaVα2δ1-induced functional modulation of the pore-forming CaV1.2 subunit"

    Article Title: A three-way inter-molecular network accounts for the CaVα2δ1-induced functional modulation of the pore-forming CaV1.2 subunit

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.001902

    Ca V β3/Ca V 1.2 protein complex coimmunoprecipitated with Ca V α2δ1 V260R. HEKT cells were transiently transfected with pCMV-Ca V 1.2 and pCMV-Ca V β3–c-Myc and either pmCherry-Ca V α2δ1-HA WT, or pmCherry-Ca V α2δ1-HA D259R, or pmCherry-Ca V α2δ1-HA V260R, or pmCherry-Ca V α2δ1-HA S261R, or pmCherry-Ca V α2δ1-HA G262R, or pmCherry-Ca V α2δ1-HA S263R. Cell lysates were immunoprecipitated ( IP ) overnight with anti-c-Myc magnetic beads to capture Ca V β3, eluted in a Laemmli buffer, and fractionated by SDS-PAGE using 8% gels. A, immunoblotting ( IB ) was carried out on total proteins (20 μg) before the immunoprecipitation assay ( input lane ) to confirm that constructs were expressed at the expected molecular weight. The signal for the housekeeping protein GAPDH is shown. B, immunoblotting was carried out after eluting the protein complexes from the beads with anti-Ca V 1.2, anti-Ca V α2δ1, and anti-Ca V β3 antibody (from top to bottom, as indicated). Images for Ca V α2δ1 were captured after shorter (1 s) or longer exposure times until the signal was saturated (20 s). All immunoblots were carried out in parallel under the same transfection and extraction conditions. These assays were successfully repeated three times with similar results.
    Figure Legend Snippet: Ca V β3/Ca V 1.2 protein complex coimmunoprecipitated with Ca V α2δ1 V260R. HEKT cells were transiently transfected with pCMV-Ca V 1.2 and pCMV-Ca V β3–c-Myc and either pmCherry-Ca V α2δ1-HA WT, or pmCherry-Ca V α2δ1-HA D259R, or pmCherry-Ca V α2δ1-HA V260R, or pmCherry-Ca V α2δ1-HA S261R, or pmCherry-Ca V α2δ1-HA G262R, or pmCherry-Ca V α2δ1-HA S263R. Cell lysates were immunoprecipitated ( IP ) overnight with anti-c-Myc magnetic beads to capture Ca V β3, eluted in a Laemmli buffer, and fractionated by SDS-PAGE using 8% gels. A, immunoblotting ( IB ) was carried out on total proteins (20 μg) before the immunoprecipitation assay ( input lane ) to confirm that constructs were expressed at the expected molecular weight. The signal for the housekeeping protein GAPDH is shown. B, immunoblotting was carried out after eluting the protein complexes from the beads with anti-Ca V 1.2, anti-Ca V α2δ1, and anti-Ca V β3 antibody (from top to bottom, as indicated). Images for Ca V α2δ1 were captured after shorter (1 s) or longer exposure times until the signal was saturated (20 s). All immunoblots were carried out in parallel under the same transfection and extraction conditions. These assays were successfully repeated three times with similar results.

    Techniques Used: Transfection, Immunoprecipitation, Magnetic Beads, SDS Page, Construct, Molecular Weight, Western Blot

    23) Product Images from "Specific Alterations in Astrocyte Properties via the GluA2-GAPDH Complex Associated with Multiple Sclerosis"

    Article Title: Specific Alterations in Astrocyte Properties via the GluA2-GAPDH Complex Associated with Multiple Sclerosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31318-4

    Disrupting GluA2-GAPDH interaction directly affects astrocytes morphology and reactivity in primary astrocyte cultures. (a ) Immunostaining of GFAP was performed on primary astrocyte cultures and fluorescent images were captured at a low magnification 10x to provide an overall view of astrocyte cultures. Scale Bar: 100 μm. (b) The percentage of GFAP + cells was measured as the number of GFAP-labeled cells per total number. There was a significantly higher proportion of reactive astrocytes with LPS and LPS with TAT-control peptide when compared to no treatment controls, but GluA2-G-Gpep treatment effectively reduced this increase (No Treatment: n = 15; LPS: n = 13; TAT-Control Pep: n = 13; GluA2-G-Gpep: n = 12 ROIs from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). (c) Higher magnification images at 60 × of individual astrocytes immunostained with GFAP were captured for morphological analyses. Scale Bar: 20 μm. (d ) There was a pronounced increase in GFAP fluorescent intensity, astrocyte surface area and the number of primary branches in LPS-stimulated astrocytes vs. control group. GluA2-G-Gpep administration decreased GFAP intensity and the number of primary processes, but not astrocyte surface area (No Treatment: n = 22; LPS: n = 23; GluA2-G-Gpep: n = 25 astrocytes from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). (e ) Western blot analysis confirmed that GFAP protein expression was higher in LPS-induced astrocytes than non-treated cells, and GluA2-G-Gpep treatment was able to revert its expression back to control levels (n = 4 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). Full-length Western blots are presented in Supplementary Fig. 4a . (f) Time-lapse imaging of primary astrocytes further revealed more processes with strong development of intermediate filaments in LPS-induced astrocytes. Consistently, GluA2-G-Gpep treatment normalized this phenotype with less filaments developed. White arrows indicate the change in astrocyte processes. Scale Bar: 20 μm. (g) Quantification of the percent change in astrocyte surface area between time 0 and 60 hours showed a significant increase with LPS treatment, but was decreased upon GluA2-G-Gpep addition (No Treatment: n = 7; LPS: n = 7; GluA2-G-Gpep: n = 6 cells from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). Data are presented as mean ± SEM. * p
    Figure Legend Snippet: Disrupting GluA2-GAPDH interaction directly affects astrocytes morphology and reactivity in primary astrocyte cultures. (a ) Immunostaining of GFAP was performed on primary astrocyte cultures and fluorescent images were captured at a low magnification 10x to provide an overall view of astrocyte cultures. Scale Bar: 100 μm. (b) The percentage of GFAP + cells was measured as the number of GFAP-labeled cells per total number. There was a significantly higher proportion of reactive astrocytes with LPS and LPS with TAT-control peptide when compared to no treatment controls, but GluA2-G-Gpep treatment effectively reduced this increase (No Treatment: n = 15; LPS: n = 13; TAT-Control Pep: n = 13; GluA2-G-Gpep: n = 12 ROIs from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). (c) Higher magnification images at 60 × of individual astrocytes immunostained with GFAP were captured for morphological analyses. Scale Bar: 20 μm. (d ) There was a pronounced increase in GFAP fluorescent intensity, astrocyte surface area and the number of primary branches in LPS-stimulated astrocytes vs. control group. GluA2-G-Gpep administration decreased GFAP intensity and the number of primary processes, but not astrocyte surface area (No Treatment: n = 22; LPS: n = 23; GluA2-G-Gpep: n = 25 astrocytes from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). (e ) Western blot analysis confirmed that GFAP protein expression was higher in LPS-induced astrocytes than non-treated cells, and GluA2-G-Gpep treatment was able to revert its expression back to control levels (n = 4 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). Full-length Western blots are presented in Supplementary Fig. 4a . (f) Time-lapse imaging of primary astrocytes further revealed more processes with strong development of intermediate filaments in LPS-induced astrocytes. Consistently, GluA2-G-Gpep treatment normalized this phenotype with less filaments developed. White arrows indicate the change in astrocyte processes. Scale Bar: 20 μm. (g) Quantification of the percent change in astrocyte surface area between time 0 and 60 hours showed a significant increase with LPS treatment, but was decreased upon GluA2-G-Gpep addition (No Treatment: n = 7; LPS: n = 7; GluA2-G-Gpep: n = 6 cells from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). Data are presented as mean ± SEM. * p

    Techniques Used: Immunostaining, Labeling, Western Blot, Expressing, Imaging

    GluA2-GAPDH disruption reverts mitochondria morphology in reactive astrocytes but had no effects on cytokine release. ( a) Representative fluorescent images showing mitochondrial morphology in primary astrocytes using mitotracker red. Higher magnification of mitochondria are shown on the right panels. LPS-treated astrocytes showed significantly smaller mitochondria, a feature of mitochondrial fission, when compared to controls. The addition of GluA2-G-Gpep reverted this abnormal phenotype with longer, elongated mitochondria morphology. Scale Bar: 20 μm (left), 5 μm (right). (b ) The average mitochondrial length was measured in each group. LPS-treated astrocytes had shorter mitochondrial length than non-treated astrocytes, while GluA2-G-Gpep treatment resulted in similar elongated mitochondria lengths as controls (n = 100 mitochondria from 3 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). (c ) Cytokine assessments in primary astrocyte cultures under different treatments revealed that IL-1β, IL-6 and TNFα were significantly increased with LPS stimulation. No change was observed with IL-10, IL-17 and IFNγ levels. Moreover, GluA2-G-Gpep had no effects on regulating the release of these specific cytokines (n = 3 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). Cytokine experiments were performed in duplicates for each sample. Data are presented as mean ± SEM. ** p
    Figure Legend Snippet: GluA2-GAPDH disruption reverts mitochondria morphology in reactive astrocytes but had no effects on cytokine release. ( a) Representative fluorescent images showing mitochondrial morphology in primary astrocytes using mitotracker red. Higher magnification of mitochondria are shown on the right panels. LPS-treated astrocytes showed significantly smaller mitochondria, a feature of mitochondrial fission, when compared to controls. The addition of GluA2-G-Gpep reverted this abnormal phenotype with longer, elongated mitochondria morphology. Scale Bar: 20 μm (left), 5 μm (right). (b ) The average mitochondrial length was measured in each group. LPS-treated astrocytes had shorter mitochondrial length than non-treated astrocytes, while GluA2-G-Gpep treatment resulted in similar elongated mitochondria lengths as controls (n = 100 mitochondria from 3 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). (c ) Cytokine assessments in primary astrocyte cultures under different treatments revealed that IL-1β, IL-6 and TNFα were significantly increased with LPS stimulation. No change was observed with IL-10, IL-17 and IFNγ levels. Moreover, GluA2-G-Gpep had no effects on regulating the release of these specific cytokines (n = 3 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). Cytokine experiments were performed in duplicates for each sample. Data are presented as mean ± SEM. ** p

    Techniques Used:

    GluA2-G-Gpep treatment reduces the elevated nuclear GAPDH, p53 and p53(S15) in LPS-stimulated astrocytes. ( a) Immunofluorescent images labeling GAPDH in primary astrocytes with different treatments. Higher magnification images are shown on the right. Astrocytes with LPS stimulation had significantly more GAPDH near nuclear membrane and within nucleus, but disruption of GluA2-GAPDH interaction with GluA2-G-Gpep prevented this effect. Scale Bar: 20 μm (left), 5 μm (right). (b) Quantification of GAPDH fluorescence intensity around the nucleus resulted in similar outcomes of higher expression in LPS-treated cells and decreased with GluA2-G-Gpep (n = 8 cells from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). (c,d ) Western blot analysis with nuclear fraction astrocyte proteins resulted in an increase of GAPDH protein levels, as well as p53 and p53 phosphorylated S15 with LPS. GluA2-G-Gpep treatment significantly reversed the expression to control levels (n = 3 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). Full-length Western blots are presented in Supplementary Fig. 5b . Data are presented as mean ± SEM. * p
    Figure Legend Snippet: GluA2-G-Gpep treatment reduces the elevated nuclear GAPDH, p53 and p53(S15) in LPS-stimulated astrocytes. ( a) Immunofluorescent images labeling GAPDH in primary astrocytes with different treatments. Higher magnification images are shown on the right. Astrocytes with LPS stimulation had significantly more GAPDH near nuclear membrane and within nucleus, but disruption of GluA2-GAPDH interaction with GluA2-G-Gpep prevented this effect. Scale Bar: 20 μm (left), 5 μm (right). (b) Quantification of GAPDH fluorescence intensity around the nucleus resulted in similar outcomes of higher expression in LPS-treated cells and decreased with GluA2-G-Gpep (n = 8 cells from 3 different cultures, one-way ANOVA followed by Bonferroni post hoc test). (c,d ) Western blot analysis with nuclear fraction astrocyte proteins resulted in an increase of GAPDH protein levels, as well as p53 and p53 phosphorylated S15 with LPS. GluA2-G-Gpep treatment significantly reversed the expression to control levels (n = 3 different cultures per group, one-way ANOVA followed by Bonferroni post hoc test). Full-length Western blots are presented in Supplementary Fig. 5b . Data are presented as mean ± SEM. * p

    Techniques Used: Labeling, Fluorescence, Expressing, Western Blot

    Disrupting GluA2-GAPDH interaction with GluA2-G-Gpep reduces astrocytes reactivity in the EAE mice. ( a ) Representative fluorescent images showing GFAP-labeled astrocytes in spinal cord sections of sham, EAE, EAE with TAT-control peptide (10 μM) and EAE with GluA2-G-Gpep (10 μM) mice. Scale Bar: 100 μm. Higher magnification images are shown below. Scale Bar: 20 μm. The analyzed regions include dorsal, intermediate and ventral grey matter. (b ) There was a significant increase in the number of GFAP + cells in the EAE and EAE with TAT-control peptide groups when compared sham controls in all spinal cord regions. GluA2-G-Gpep treatment produced a marked reduction in GFAP-labeled cell numbers comparable to controls. ( c ) Astrocyte reactivity was quantified using mean grey values of fluorescent intensity, and measured as percent area occupancy from a normalized thresholding scale with ImageJ. EAE and EAE + TAT-control pep mice had significantly higher fluorescence intensity values and fluorescent occupancy vs. sham animals, while administration of GluA2-G-Gpep reduced both parameters (Sham: n = 12; EAE: n = 10; TAT-Control Pep: n = 16; GluA2-G-Gpep: n = 13 sections from 3 different spinal cords, two-way ANOVA followed by Bonferroni post hoc test). (d) The changes in GFAP expression among the different groups were confirmed with Western blot experiments (n = 4 spinal cords per group, one-way ANOVA followed by Bonferroni post hoc test). The full-length blot is shown and quantification of protein expression was normalized with actin loading controls and expressed as a percentage of sham groups. Data are presented as mean ± SEM. * p
    Figure Legend Snippet: Disrupting GluA2-GAPDH interaction with GluA2-G-Gpep reduces astrocytes reactivity in the EAE mice. ( a ) Representative fluorescent images showing GFAP-labeled astrocytes in spinal cord sections of sham, EAE, EAE with TAT-control peptide (10 μM) and EAE with GluA2-G-Gpep (10 μM) mice. Scale Bar: 100 μm. Higher magnification images are shown below. Scale Bar: 20 μm. The analyzed regions include dorsal, intermediate and ventral grey matter. (b ) There was a significant increase in the number of GFAP + cells in the EAE and EAE with TAT-control peptide groups when compared sham controls in all spinal cord regions. GluA2-G-Gpep treatment produced a marked reduction in GFAP-labeled cell numbers comparable to controls. ( c ) Astrocyte reactivity was quantified using mean grey values of fluorescent intensity, and measured as percent area occupancy from a normalized thresholding scale with ImageJ. EAE and EAE + TAT-control pep mice had significantly higher fluorescence intensity values and fluorescent occupancy vs. sham animals, while administration of GluA2-G-Gpep reduced both parameters (Sham: n = 12; EAE: n = 10; TAT-Control Pep: n = 16; GluA2-G-Gpep: n = 13 sections from 3 different spinal cords, two-way ANOVA followed by Bonferroni post hoc test). (d) The changes in GFAP expression among the different groups were confirmed with Western blot experiments (n = 4 spinal cords per group, one-way ANOVA followed by Bonferroni post hoc test). The full-length blot is shown and quantification of protein expression was normalized with actin loading controls and expressed as a percentage of sham groups. Data are presented as mean ± SEM. * p

    Techniques Used: Mouse Assay, Labeling, Produced, Fluorescence, Expressing, Western Blot

    24) Product Images from "FoxP1 orchestration of ASD-relevant signaling pathways in the striatum"

    Article Title: FoxP1 orchestration of ASD-relevant signaling pathways in the striatum

    Journal: Genes & Development

    doi: 10.1101/gad.267989.115

    Regulation of ASD genes by Foxp1 in the mouse brain. ( A ) Representative immunoblot displaying reduced Foxp1 protein levels in the hippocampus (HIP) and striatum (STR), but not the neocortex (CTX), of Foxp1 +/− mice. Gapdh was used as a loading control. ( B ) Quantification of Foxp1 expression in adult Foxp1 +/− mouse brains. Data are represented as means ± SEM. n = 4 mice per genotype for each region. (*) P = 0.033 (hippocampus); (*) P = 0.0163 (striatum), Student's t -test, compared with wild-type levels normalized to Gapdh. ( C ) Venn diagram showing overlaps between the differentially expressed genes (DEGs) in the mouse and ASD gene lists (144 genes between the hippocampus and striatum [ P = 1.21 × 10 −26 ], 116 genes between the hippocampus and ASD [ P = 3.74 × 10 −9 ], and 43 genes between the striatum and ASD [ P = 0.002], hypergeometric test [ P -values were adjusted using Benjamini-Hochberg FDR procedure]). ( D ) Confirmation of salient ASD-related gene targets in independent striatal samples from Foxp1 +/− mice using quantitative RT–PCR (qRT–PCR). Data are represented as means ± SEM. n = 4 mice per genotype. With the exception of Dner , all qRT–PCR values displayed are significant at P
    Figure Legend Snippet: Regulation of ASD genes by Foxp1 in the mouse brain. ( A ) Representative immunoblot displaying reduced Foxp1 protein levels in the hippocampus (HIP) and striatum (STR), but not the neocortex (CTX), of Foxp1 +/− mice. Gapdh was used as a loading control. ( B ) Quantification of Foxp1 expression in adult Foxp1 +/− mouse brains. Data are represented as means ± SEM. n = 4 mice per genotype for each region. (*) P = 0.033 (hippocampus); (*) P = 0.0163 (striatum), Student's t -test, compared with wild-type levels normalized to Gapdh. ( C ) Venn diagram showing overlaps between the differentially expressed genes (DEGs) in the mouse and ASD gene lists (144 genes between the hippocampus and striatum [ P = 1.21 × 10 −26 ], 116 genes between the hippocampus and ASD [ P = 3.74 × 10 −9 ], and 43 genes between the striatum and ASD [ P = 0.002], hypergeometric test [ P -values were adjusted using Benjamini-Hochberg FDR procedure]). ( D ) Confirmation of salient ASD-related gene targets in independent striatal samples from Foxp1 +/− mice using quantitative RT–PCR (qRT–PCR). Data are represented as means ± SEM. n = 4 mice per genotype. With the exception of Dner , all qRT–PCR values displayed are significant at P

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

    25) Product Images from "ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers"

    Article Title: ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177649

    TGF-β signaling pathway is induced in gastrocnemius muscle from symptomatic hSOD1 G93A mice. (A) TGF-β (1, 2, 3), CTGF/CCN2 was detected by western-blot in protein extracts under non-reducing condition from wild-type (60 days old), pre-symptomatic (60 days old) hSOD1 G93A age-matched mice, wild-type (120 days old) and symptomatic (120 days old) hSOD1 G93A age-matched mice. GAPDH protein levels are shown as loading control. (B-C) Protein levels of TGF-β (1, 2, 3) and CTGF/CCN2 were quantified using densitometric analysis. Values correspond to the mean ± SEM of four animals for each experimental condition. One-way ANOVA, *** p
    Figure Legend Snippet: TGF-β signaling pathway is induced in gastrocnemius muscle from symptomatic hSOD1 G93A mice. (A) TGF-β (1, 2, 3), CTGF/CCN2 was detected by western-blot in protein extracts under non-reducing condition from wild-type (60 days old), pre-symptomatic (60 days old) hSOD1 G93A age-matched mice, wild-type (120 days old) and symptomatic (120 days old) hSOD1 G93A age-matched mice. GAPDH protein levels are shown as loading control. (B-C) Protein levels of TGF-β (1, 2, 3) and CTGF/CCN2 were quantified using densitometric analysis. Values correspond to the mean ± SEM of four animals for each experimental condition. One-way ANOVA, *** p

    Techniques Used: Mouse Assay, Western Blot

    26) Product Images from "Development of an oncolytic Herpes Simplex Virus using a tumor-specific HIF-responsive promoter"

    Article Title: Development of an oncolytic Herpes Simplex Virus using a tumor-specific HIF-responsive promoter

    Journal: Cancer gene therapy

    doi: 10.1038/cgt.2010.62

    ICP4 (34kDa) and thymidine kinase (TK) expression by HIF-E6L-HSV and HIF-V6R-HSV E5 cells were infected at MOI 0.25 with the indicated (a) HIF-E6L-HSV (clones 1-5) or (b) HIF-V6R-HSV (clones 7, 9, 16, 18, 21, 8) and then grown under normoxia. As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 30μg of total cell lysate was examined by immunoblotting for the presence of full length ICP4 (175 kDa), truncated ICP4 (34kDa), TK (40 kDa), and GAPDH (loading control, 36 kDa) protein expression. The ICP4 bands are shown at two different exposures to better visualize the 175 kDa and 34 kDa ICP4 proteins. Asterisks indicate a non-specific band that is cross-reactive with the anti-ICP4 antibody.
    Figure Legend Snippet: ICP4 (34kDa) and thymidine kinase (TK) expression by HIF-E6L-HSV and HIF-V6R-HSV E5 cells were infected at MOI 0.25 with the indicated (a) HIF-E6L-HSV (clones 1-5) or (b) HIF-V6R-HSV (clones 7, 9, 16, 18, 21, 8) and then grown under normoxia. As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 30μg of total cell lysate was examined by immunoblotting for the presence of full length ICP4 (175 kDa), truncated ICP4 (34kDa), TK (40 kDa), and GAPDH (loading control, 36 kDa) protein expression. The ICP4 bands are shown at two different exposures to better visualize the 175 kDa and 34 kDa ICP4 proteins. Asterisks indicate a non-specific band that is cross-reactive with the anti-ICP4 antibody.

    Techniques Used: Expressing, Infection

    Strains HIF-E6L-HSV and HIF-V6R-HSV express full length ICP4 (175 kDa) and expression is not HIF-dependent LN229 cells were infected at MOI 0.05 with the indicated HIF-E6L-HSV (clones 1-5) or HIF-V6R-HSV (clones 6-22) clone and then grown under normoxia (N) or hypoxia (H). As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 20μg of total cell lysate was western blotted for full length ICP4 (175 kDa) protein expression. GAPDH (36 kDa) or actin (42 kDa) protein expression was used as a loading control. M=molecular weight standard lane.
    Figure Legend Snippet: Strains HIF-E6L-HSV and HIF-V6R-HSV express full length ICP4 (175 kDa) and expression is not HIF-dependent LN229 cells were infected at MOI 0.05 with the indicated HIF-E6L-HSV (clones 1-5) or HIF-V6R-HSV (clones 6-22) clone and then grown under normoxia (N) or hypoxia (H). As controls, cells were mock- or virus-infected with HSV-1 strain KOS or d120. One day later, 20μg of total cell lysate was western blotted for full length ICP4 (175 kDa) protein expression. GAPDH (36 kDa) or actin (42 kDa) protein expression was used as a loading control. M=molecular weight standard lane.

    Techniques Used: Expressing, Infection, Western Blot, Molecular Weight

    27) Product Images from "A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity"

    Article Title: A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity

    Journal: Nature immunology

    doi: 10.1038/ni.3393

    NS3 inhibits binding of RIG-I to 14-3-3ε, preventing the translocation of activated RIG-I to mitochondria ( a ) Ubiquitination of RIG-I-FLAG in transfected HEK293T cells that were mock-infected or infected with SeV (50 HAU/ml) for 19 h. WCLs were subjected to FLAG-PD, followed by IB with anti-ubiquitin (Ub) and anti-FLAG. ( b ) Ubiquitination of endogenous RIG-I in Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-Ub and anti-RIG-I. ( c ) Binding of endogenous RIG-I, TRIM25 and 14-3-3ε in transfected HEK293T cells that were infected with SeV (50 HAU/ml) for 23 h. WCLs were subjected to IP with anti-RIG-I (left) or anti-TRIM25 (right), followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. The data shown are from the same experiment. ( d ) Huh7 cells were mock-infected, or infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. ( e ) HEK293T cells were transfected with RIG-I-FLAG together with vector or increasing amounts of GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h, and FLAG-PD was performed. ( f ) Cytosol-mitochondria fractionation of WCLs from Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 22 h. IB was performed with anti-RIG-I, anti-MAVS and anti-GAPDH. RIG-I and NS3 expressions were determined in the WCL. Data are representative of at least 2 independent experiments ( a–f ).
    Figure Legend Snippet: NS3 inhibits binding of RIG-I to 14-3-3ε, preventing the translocation of activated RIG-I to mitochondria ( a ) Ubiquitination of RIG-I-FLAG in transfected HEK293T cells that were mock-infected or infected with SeV (50 HAU/ml) for 19 h. WCLs were subjected to FLAG-PD, followed by IB with anti-ubiquitin (Ub) and anti-FLAG. ( b ) Ubiquitination of endogenous RIG-I in Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-Ub and anti-RIG-I. ( c ) Binding of endogenous RIG-I, TRIM25 and 14-3-3ε in transfected HEK293T cells that were infected with SeV (50 HAU/ml) for 23 h. WCLs were subjected to IP with anti-RIG-I (left) or anti-TRIM25 (right), followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. The data shown are from the same experiment. ( d ) Huh7 cells were mock-infected, or infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 18 h. IP with anti-RIG-I was performed, followed by IB with anti-14-3-3ε, anti-TRIM25 or anti-RIG-I. ( e ) HEK293T cells were transfected with RIG-I-FLAG together with vector or increasing amounts of GST-NS3. 48 h later, cells were infected with SeV (50 HAU/ml) for 20 h, and FLAG-PD was performed. ( f ) Cytosol-mitochondria fractionation of WCLs from Huh7 cells that were mock-infected, infected with DV2 NGC (MOI 1) or SeV (50 HAU/ml) for 22 h. IB was performed with anti-RIG-I, anti-MAVS and anti-GAPDH. RIG-I and NS3 expressions were determined in the WCL. Data are representative of at least 2 independent experiments ( a–f ).

    Techniques Used: Binding Assay, Translocation Assay, Transfection, Infection, Plasmid Preparation, Fractionation

    28) Product Images from "RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells"

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18851-4

    Effects of RGS7 mutation on RGS7 stability and activity. ( a ) The Human RGS7 protein, with conserved domains indicated as blocks, including the Dishevelled domain (DEP); G Protein Gamma-like domain (GGL); RGS domain (RGS). Somatic mutations indicated with arrows. Red triangles indicate deleterious mutations. ( b ) Three dimensional structure of RGS7 N-Terminus, predicting that R44 is involved in an H-bond network with S58 and D61. ( c ) Cells expressing wild-type or mutant RGS7 were treated with cycloheximide (CHX), collected at different time points and then immunoblotted with anti-FLAG antibody. Anti-Cyclin D1 was used as a control and anti-GAPDH was used for normalization. ( d ) A Schematic representation of the BRET-based assay to monitor G protein signaling cycle. Activation of the D2R causes the G protein heterotrimer to dissociate into Gα and Gβγ subunits. Released Gβγ subunits tagged with Venus fluorescent protein interacts with Nluc–tagged reporter G protein receptor kinase (GRK) to produce the BRET signal. Upon termination of D2R activation by antagonist haloperidol, Gαo subunit hydrolyses GTP and reassociates with Gβγ subunits, quenching the BRET signal. ( e ) Time course of normalized BRET responses recorded in a representative experiment. Left . The deactivation phase after antagonist application is shown. Wild-type RGS7 or mutant were transfected at equal amount of cDNA (210 ng) together with dopamine D2 receptor, Gαo, and BRET sensor pair. Right . Quantification of the exponential decay kinetics of the response. BRET values were averaged from four or six replicates. *P
    Figure Legend Snippet: Effects of RGS7 mutation on RGS7 stability and activity. ( a ) The Human RGS7 protein, with conserved domains indicated as blocks, including the Dishevelled domain (DEP); G Protein Gamma-like domain (GGL); RGS domain (RGS). Somatic mutations indicated with arrows. Red triangles indicate deleterious mutations. ( b ) Three dimensional structure of RGS7 N-Terminus, predicting that R44 is involved in an H-bond network with S58 and D61. ( c ) Cells expressing wild-type or mutant RGS7 were treated with cycloheximide (CHX), collected at different time points and then immunoblotted with anti-FLAG antibody. Anti-Cyclin D1 was used as a control and anti-GAPDH was used for normalization. ( d ) A Schematic representation of the BRET-based assay to monitor G protein signaling cycle. Activation of the D2R causes the G protein heterotrimer to dissociate into Gα and Gβγ subunits. Released Gβγ subunits tagged with Venus fluorescent protein interacts with Nluc–tagged reporter G protein receptor kinase (GRK) to produce the BRET signal. Upon termination of D2R activation by antagonist haloperidol, Gαo subunit hydrolyses GTP and reassociates with Gβγ subunits, quenching the BRET signal. ( e ) Time course of normalized BRET responses recorded in a representative experiment. Left . The deactivation phase after antagonist application is shown. Wild-type RGS7 or mutant were transfected at equal amount of cDNA (210 ng) together with dopamine D2 receptor, Gαo, and BRET sensor pair. Right . Quantification of the exponential decay kinetics of the response. BRET values were averaged from four or six replicates. *P

    Techniques Used: Mutagenesis, Activity Assay, Expressing, Bioluminescence Resonance Energy Transfer, Activation Assay, Transfection

    29) Product Images from "Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase Cι to Associate with Microtubules and to Recruit Dynein"

    Article Title: Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase Cι to Associate with Microtubules and to Recruit Dynein

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807756200

    Rab2 requires GAPDH and PKC ι to associate with MTs. A , pure bovine brain tubulin (1 μg) was preassembled as outlined under “Experimental Procedures” and then incubated with Rab2 (200 ng), aPKCι (200 ng), or GAPDH
    Figure Legend Snippet: Rab2 requires GAPDH and PKC ι to associate with MTs. A , pure bovine brain tubulin (1 μg) was preassembled as outlined under “Experimental Procedures” and then incubated with Rab2 (200 ng), aPKCι (200 ng), or GAPDH

    Techniques Used: Incubation

    30) Product Images from "A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency"

    Article Title: A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Journal: Scientific Reports

    doi: 10.1038/srep41408

    DMSA-SPIONs accumulate in endosomal compartments with increasing chase periods while aminolipid-SPIONs remain tethered at the cell surface. ( A ) Quantitative immunoblot analysis (equal protein loading) for the indicated organelle marker proteins in isolated fractions using DMSA-SPIONs with increasing chase periods of ( A ) 0 min, ( B ) 1 h, ( C ) 2 h, ( D ) 3 h, ( E ) 4 h or in fractions isolated using aminolipid-SPIONs ( F ) as a fold increase relative to total cell lysate (mean ± SEM, n = 3). Na + K + (Na + K + -ATPase) is a PM-localized integral membrane protein, EEA1 marks early endosomes, Rab7 late endosomes and Lamp1 lysosomes. RIB (Ribophorin) and GAPDH represent endoplasmic reticulum and cytosol, respectively. TCL - total cell lysate; UB - Unbound/non-magnetic fraction and B - Bound/magnetic fraction. ( G ) Silver staining of total cell lysate (TCL), bound/magnetic fraction isolated using SPIONs functionalized with DMSA (LYS) or with aminolipids (PM). The distinct protein profile in the bound fraction as observed by the lane scan underscores the enrichment of specific protein subsets (M - Marker, SeeBlue plus2 rainbow protein marker (Invitrogen)). TEM analysis of the fractions isolated using DMSA- ( H ) and aminolipid- ( I ) coated SPIONs. Scale bar = 0.5 μm.
    Figure Legend Snippet: DMSA-SPIONs accumulate in endosomal compartments with increasing chase periods while aminolipid-SPIONs remain tethered at the cell surface. ( A ) Quantitative immunoblot analysis (equal protein loading) for the indicated organelle marker proteins in isolated fractions using DMSA-SPIONs with increasing chase periods of ( A ) 0 min, ( B ) 1 h, ( C ) 2 h, ( D ) 3 h, ( E ) 4 h or in fractions isolated using aminolipid-SPIONs ( F ) as a fold increase relative to total cell lysate (mean ± SEM, n = 3). Na + K + (Na + K + -ATPase) is a PM-localized integral membrane protein, EEA1 marks early endosomes, Rab7 late endosomes and Lamp1 lysosomes. RIB (Ribophorin) and GAPDH represent endoplasmic reticulum and cytosol, respectively. TCL - total cell lysate; UB - Unbound/non-magnetic fraction and B - Bound/magnetic fraction. ( G ) Silver staining of total cell lysate (TCL), bound/magnetic fraction isolated using SPIONs functionalized with DMSA (LYS) or with aminolipids (PM). The distinct protein profile in the bound fraction as observed by the lane scan underscores the enrichment of specific protein subsets (M - Marker, SeeBlue plus2 rainbow protein marker (Invitrogen)). TEM analysis of the fractions isolated using DMSA- ( H ) and aminolipid- ( I ) coated SPIONs. Scale bar = 0.5 μm.

    Techniques Used: Marker, Isolation, Silver Staining, Transmission Electron Microscopy

    31) Product Images from "Mild Functional Differences of Dynamin 2 Mutations Associated to Centronuclear Myopathy and Charcot-Marie-Tooth Peripheral Neuropathy"

    Article Title: Mild Functional Differences of Dynamin 2 Mutations Associated to Centronuclear Myopathy and Charcot-Marie-Tooth Peripheral Neuropathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027498

    MT dynamics in CNM patient fibroblasts. (A) MT dynamics in fibroblasts from control and CNM patients harboring the R465W or S619L mutation. Fibroblasts were left untreated or were treated with 5 µM nocodazole for 2 h at 37°C. To allow for partial recovery of MTs, nocodazole was removed and cells were placed at 37°C for 5 min. To view MTs, the cells were stained with β tubulin specific antibodies. Stabilized MTs were visualized with acetylated tubulin-specific antibodies. ( B ) Levels of acetylated tubulin are comparable for control and patient fibroblast lines. Equal amounts of indicated fibroblast lysates were subjected to SDS-PAGE and were analyzed by western blot employing anti-acetylated tubulin antibodies and controls (anti-EEA1, anti-dynamin 2 and anti-GAPDH).
    Figure Legend Snippet: MT dynamics in CNM patient fibroblasts. (A) MT dynamics in fibroblasts from control and CNM patients harboring the R465W or S619L mutation. Fibroblasts were left untreated or were treated with 5 µM nocodazole for 2 h at 37°C. To allow for partial recovery of MTs, nocodazole was removed and cells were placed at 37°C for 5 min. To view MTs, the cells were stained with β tubulin specific antibodies. Stabilized MTs were visualized with acetylated tubulin-specific antibodies. ( B ) Levels of acetylated tubulin are comparable for control and patient fibroblast lines. Equal amounts of indicated fibroblast lysates were subjected to SDS-PAGE and were analyzed by western blot employing anti-acetylated tubulin antibodies and controls (anti-EEA1, anti-dynamin 2 and anti-GAPDH).

    Techniques Used: Mutagenesis, Staining, SDS Page, Western Blot

    32) Product Images from "Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma"

    Article Title: Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma

    Journal: Biomolecules

    doi: 10.3390/biom9100622

    shRNA-mediated knockdown of caveolin-1 expression of 1321N1 astrocytoma cells. ( A ) Immunoblot analysis of hHAP2Y 2 R, caveolin-1 (Cav-1), and GAPDH (control) expression in serum-starved wild-type (WT) 1321N1 cells (lane 1), human 1321N1 cells expressing hHAP2Y 2 R (hHAP2Y 2 R 1321N1 cells) (lane 3), or cells infected with Cav-1 shRNA lentiviral particles (lanes 2 and 4; Cav-1 knockdown (KD)). ( B ) Densitometric analysis of immunoblots indicates the level of Cav-1 normalized to GAPDH expression. Results are presented as the means ± S.E.M. (n = 3; *** p
    Figure Legend Snippet: shRNA-mediated knockdown of caveolin-1 expression of 1321N1 astrocytoma cells. ( A ) Immunoblot analysis of hHAP2Y 2 R, caveolin-1 (Cav-1), and GAPDH (control) expression in serum-starved wild-type (WT) 1321N1 cells (lane 1), human 1321N1 cells expressing hHAP2Y 2 R (hHAP2Y 2 R 1321N1 cells) (lane 3), or cells infected with Cav-1 shRNA lentiviral particles (lanes 2 and 4; Cav-1 knockdown (KD)). ( B ) Densitometric analysis of immunoblots indicates the level of Cav-1 normalized to GAPDH expression. Results are presented as the means ± S.E.M. (n = 3; *** p

    Techniques Used: shRNA, Expressing, Infection, Western Blot

    33) Product Images from "Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy"

    Article Title: Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy

    Journal: Nature communications

    doi: 10.1038/ncomms5425

    Inhibition of Nox2-activity reduces oxidative stress and Src kinase-mediated impaired autophagy ( a ) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalse. ( b ) Measurement of intracellular glutathione redox potential with Grx1-roGFP2. ( c ) Analysis of Rac1 and ( d ) Src. ( e ) Immunoblot of precipitated p47 phox probed with an anti-phosphoserine or anti-p47 phox antibody. ( f ) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor. ( g ) Extracellular ROS production was assessed using Amplex-red dye. ( h ) Plasma membrane calcium influx was measured by analyzing the Fura-2 fluorescence quench rate upon addition of extracellular Mn 2+ . ( i ) Intracellular RNS generation was measured using DAF-FM. Bars represent average ±SEM from n=15 individual fibers for each condition in ( a , b , f , g , j and i ). Markers of autophagy were analyzed in isolated fibers (incubated with or without PP2) from FDBs. ( k ) Autophagosome formation was analyzed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. ( l ) Confocal microscopy detected p62-LC3 localization in single fibers from FDBs (scale bar=140 μm and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±SEM from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey’s post-hoc test. *p
    Figure Legend Snippet: Inhibition of Nox2-activity reduces oxidative stress and Src kinase-mediated impaired autophagy ( a ) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalse. ( b ) Measurement of intracellular glutathione redox potential with Grx1-roGFP2. ( c ) Analysis of Rac1 and ( d ) Src. ( e ) Immunoblot of precipitated p47 phox probed with an anti-phosphoserine or anti-p47 phox antibody. ( f ) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor. ( g ) Extracellular ROS production was assessed using Amplex-red dye. ( h ) Plasma membrane calcium influx was measured by analyzing the Fura-2 fluorescence quench rate upon addition of extracellular Mn 2+ . ( i ) Intracellular RNS generation was measured using DAF-FM. Bars represent average ±SEM from n=15 individual fibers for each condition in ( a , b , f , g , j and i ). Markers of autophagy were analyzed in isolated fibers (incubated with or without PP2) from FDBs. ( k ) Autophagosome formation was analyzed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. ( l ) Confocal microscopy detected p62-LC3 localization in single fibers from FDBs (scale bar=140 μm and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±SEM from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey’s post-hoc test. *p

    Techniques Used: Inhibition, Activity Assay, Fluorescence, Isolation, Incubation, Microscopy, Confocal Microscopy, Western Blot

    34) Product Images from "Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells"

    Article Title: Extracellular ?-Synuclein Leads to Microtubule Destabilization via GSK-3?-Dependent Tau Phosphorylation in PC12 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094259

    The effect of ASN on the protein level and gene expression for GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The total level of GSK-3β was determined using Western blot analysis. ( A ) Immunoreactivity of GSK-3β and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of GSK-3β immunoreactivity. Results were normalized to GAPDH levels. ( C ) The gene expression for total GSK-3β was measured with real-time PCR. Data represent the mean value ± S.E.M. for 5 independent experiments. * p
    Figure Legend Snippet: The effect of ASN on the protein level and gene expression for GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The total level of GSK-3β was determined using Western blot analysis. ( A ) Immunoreactivity of GSK-3β and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of GSK-3β immunoreactivity. Results were normalized to GAPDH levels. ( C ) The gene expression for total GSK-3β was measured with real-time PCR. Data represent the mean value ± S.E.M. for 5 independent experiments. * p

    Techniques Used: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction

    The effect of CDK5 and GSK-3β inhibitors on ASN-evoked increase in Tau phosphorylation. PC12 cells were incubated with 10 μM ASN in the presence of 10 μM inhibitors for 48 h. BML-259 and SB-216763 were used as CDK5 and GSK-3β inhibitor, respectively. The level of Tau phosphorylation at Ser396 was determined using the Western blotting method. ( A ) Immunoreactivity of p-Tau (Ser396) and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of p-Tau (Ser396) immunoreactivity. Results were normalized to GAPDH levels. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p
    Figure Legend Snippet: The effect of CDK5 and GSK-3β inhibitors on ASN-evoked increase in Tau phosphorylation. PC12 cells were incubated with 10 μM ASN in the presence of 10 μM inhibitors for 48 h. BML-259 and SB-216763 were used as CDK5 and GSK-3β inhibitor, respectively. The level of Tau phosphorylation at Ser396 was determined using the Western blotting method. ( A ) Immunoreactivity of p-Tau (Ser396) and GAPDH protein, which is presented as a loading control. ( B ) Densitometric analysis of p-Tau (Ser396) immunoreactivity. Results were normalized to GAPDH levels. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p

    Techniques Used: Incubation, Western Blot

    The effect of ASN on the phosphorylation state of GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The phosphorylation status of GSK-3β at Ser9 and Tyr216 was determined using Western blot analysis. ( A ) Immunoreactivity of p-GSK-3β (Ser9). ( B ) Immunoreactivity of p-GSK-3β (Tyr216). GAPDH was used as a loading control. Data represent the mean value ± S.E.M. for 5 independent experiments. * p
    Figure Legend Snippet: The effect of ASN on the phosphorylation state of GSK-3β. PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The phosphorylation status of GSK-3β at Ser9 and Tyr216 was determined using Western blot analysis. ( A ) Immunoreactivity of p-GSK-3β (Ser9). ( B ) Immunoreactivity of p-GSK-3β (Tyr216). GAPDH was used as a loading control. Data represent the mean value ± S.E.M. for 5 independent experiments. * p

    Techniques Used: Incubation, Western Blot

    35) Product Images from "Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP, et al. Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP"

    Article Title: Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP, et al. Exosomes secreted by chronic hepatitis B patients with PNALT and liver inflammation grade ≥ A2 promoted the progression of liver cancer by transferring miR‐25‐3p to inhibit the co‐expression of TCF21 and HHIP

    Journal: Cell Proliferation

    doi: 10.1111/cpr.12833

    Both TCF21 and HHIP were target genes of miR‐25‐3p. A, The mRNA expression of TCF21 and HHIP in CHB patients with PNALT (≥A2) was detected by RT‐PCR assay. B, Co‐immunoprecipitation experiments indicated that TCF21 directly interact with HHIP in HepG2.2.15 cells. C, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in TCF21 inhibition or overexpression transfected HepG2.2.15 cells. D, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in HHIP inhibition or overexpression transfected HepG2.2.15 cells. E, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 inhibition or HHIP inhibition transfected HepG2.2.15 cells. F, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 overexpression or HHIP overexpression transfected HepG2.2.15 cells. G, TargetScan database showed that binding site of TCF21 or HHIP and miR‐340‐5p. H, Luciferase reporter assays were used to prove that miR‐340‐5p can target TCF21 or HHIP. I, J, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in miR‐25‐3p mimics or miR‐25‐3p inhibitor‐transfected HepG2.2.15 cells. K, Correlation analysis of TCF21/HHIP and miR‐25‐3p in CHB patients with PNALT (≥A2), correlation analysis of TCF21 and HHIP/miR‐25‐3p in HBV‐positive patients with liver cancer and correlation analysis of TCF21 and HHIP/miR‐25‐3p in CHB patients with PNALT (≥A2). GAPDH or U6 was used as a load control. Data are presented as the mean ± standard deviation. ** P
    Figure Legend Snippet: Both TCF21 and HHIP were target genes of miR‐25‐3p. A, The mRNA expression of TCF21 and HHIP in CHB patients with PNALT (≥A2) was detected by RT‐PCR assay. B, Co‐immunoprecipitation experiments indicated that TCF21 directly interact with HHIP in HepG2.2.15 cells. C, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in TCF21 inhibition or overexpression transfected HepG2.2.15 cells. D, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in HHIP inhibition or overexpression transfected HepG2.2.15 cells. E, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 inhibition or HHIP inhibition transfected HepG2.2.15 cells. F, The protein expression of TCF21 and HHIP was detected by Western blot assay in TCF21 overexpression or HHIP overexpression transfected HepG2.2.15 cells. G, TargetScan database showed that binding site of TCF21 or HHIP and miR‐340‐5p. H, Luciferase reporter assays were used to prove that miR‐340‐5p can target TCF21 or HHIP. I, J, The mRNA expression of TCF21 and HHIP was detected by RT‐PCR assay in miR‐25‐3p mimics or miR‐25‐3p inhibitor‐transfected HepG2.2.15 cells. K, Correlation analysis of TCF21/HHIP and miR‐25‐3p in CHB patients with PNALT (≥A2), correlation analysis of TCF21 and HHIP/miR‐25‐3p in HBV‐positive patients with liver cancer and correlation analysis of TCF21 and HHIP/miR‐25‐3p in CHB patients with PNALT (≥A2). GAPDH or U6 was used as a load control. Data are presented as the mean ± standard deviation. ** P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Inhibition, Over Expression, Transfection, Western Blot, Binding Assay, Luciferase, Standard Deviation

    The functions of miR‐25‐3p inhibitors abolishing the effects of CHB‐PNALT‐Exo (≥A2) on the proliferation and metastasis were reversed by knockdown of TCF21 and HHIP in HepG2.2.15 cells. After siRNA‐TCF21 and siRNA‐HHIP were transfected into CHB‐PNALT‐Exo (≥A2) containing miR‐25‐3p inhibitor‐treated HepG2.2.15 cells, cell viability, apoptosis, invasion, migration and the expression of cleaved caspase‐3/‐9, Ki67 and E‐cadherin were detected by CCK‐8 assay (A), flow cytometry analysis (B), clone formation assay (C), transwell assay (D), scratch‐wound assay (E) and Western blot assay (F). GAPDH was used as a load control. Data are presented as the mean ± standard deviation. ** P
    Figure Legend Snippet: The functions of miR‐25‐3p inhibitors abolishing the effects of CHB‐PNALT‐Exo (≥A2) on the proliferation and metastasis were reversed by knockdown of TCF21 and HHIP in HepG2.2.15 cells. After siRNA‐TCF21 and siRNA‐HHIP were transfected into CHB‐PNALT‐Exo (≥A2) containing miR‐25‐3p inhibitor‐treated HepG2.2.15 cells, cell viability, apoptosis, invasion, migration and the expression of cleaved caspase‐3/‐9, Ki67 and E‐cadherin were detected by CCK‐8 assay (A), flow cytometry analysis (B), clone formation assay (C), transwell assay (D), scratch‐wound assay (E) and Western blot assay (F). GAPDH was used as a load control. Data are presented as the mean ± standard deviation. ** P

    Techniques Used: Transfection, Migration, Expressing, CCK-8 Assay, Flow Cytometry, Tube Formation Assay, Transwell Assay, Scratch Wound Assay Assay, Western Blot, Standard Deviation

    36) Product Images from "Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells *"

    Article Title: Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.730226

    shRNA-mediated knockdown of caveolin-1 expression causes redistribution of the P2Y 2 R from membrane rafts. A, immunoblot analysis of hHAP2Y 2 R, Cav-1, and GAPDH (control) expression in serum-starved WT 1321N1 cells ( lane 1 ) or hHAP2Y 2 R 1321N1 cells ( lanes
    Figure Legend Snippet: shRNA-mediated knockdown of caveolin-1 expression causes redistribution of the P2Y 2 R from membrane rafts. A, immunoblot analysis of hHAP2Y 2 R, Cav-1, and GAPDH (control) expression in serum-starved WT 1321N1 cells ( lane 1 ) or hHAP2Y 2 R 1321N1 cells ( lanes

    Techniques Used: shRNA, Expressing

    Caveolin-1 knockdown inhibits P2Y 2 R-mediated increases in [Ca 2+ ] i in C6 glioma cells. A, representative immunoblot of P2Y 2 R, Cav-1, and GAPDH (control) expression in WT C6 cells ( 1st lane ), C6 cells after infection with scrambled (SCRAM) shRNA ( 2nd lane
    Figure Legend Snippet: Caveolin-1 knockdown inhibits P2Y 2 R-mediated increases in [Ca 2+ ] i in C6 glioma cells. A, representative immunoblot of P2Y 2 R, Cav-1, and GAPDH (control) expression in WT C6 cells ( 1st lane ), C6 cells after infection with scrambled (SCRAM) shRNA ( 2nd lane

    Techniques Used: Expressing, Infection, shRNA

    37) Product Images from "SUMO2/3 conjugation is an endogenous neuroprotective mechanism"

    Article Title: SUMO2/3 conjugation is an endogenous neuroprotective mechanism

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1038/jcbfm.2011.112

    Neuronal specificity and efficacy of RNA interference with SUMO2/3 and its effects on survival of mouse primary cortical neurons. At days in vitro (DIV) 3, primary cortical neurons were transduced with lentiviral particles expressing EGFP as a reporter and either SUMO2/3 or control microRNA driven by the neuron-specific synapsin promoter. ( A ) Verification of transduction efficacy and neuronal specificity of cultures transduced with lentiviral particles driven by the synapsin promoter. Immunohistochemistry was performed after paraformaldehyde (PFA) fixation on DIV 12 with antibodies against EGFP, microtubuli-associated protein 2 (MAP2), and nuclear counterstain with DAPI. The multiplicity of infection (MOI) was ∼50. Scale bar=100 μ m. ( B ) Verification of knockdown efficiency of SUMO2/3 versus control microRNAs (1=LacZ, 2=non-targeting scrambled) with and without OGD (45 minutes and 3 hours reoxygenation) shown by a representative western blot analysis. Neuronal cultures were analyzed on DIV 12 and subjected to SDS-PAGE. Membranes were probed with antibodies against SUMO2/3, EGFP, and GAPDH. EGFP expression corresponded to an equal MOI of lentiviral particles and concomitant microRNA expression. GAPDH served as a housekeeping protein and equal loading control. ( C ) SUMO2/3 microRNA does not influence baseline survival over time up to DIV 12. In brief, microscopic pictures of EGFP fluorescence (indicative for microRNA delivery) were taken at DIV 6, 9, and 12 as described in the ‘Materials and methods' section. EGFP-expressing neurons were counted and ratios were calculated for DIV 9/6 (indicated in blue) and DIV 12/9 (red) to evaluate cell survival over time. We assumed an effect size > 0.15 and performed a prospective power analysis with α =0.05 and β =0.20. There was no significant difference between groups in a two-way repeated-measures ANOVA followed by Tukey's post hoc analysis. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP2, microtubule-associated protein 2; OGD, oxygen–glucose deprivation; SUMO2/3, small ubiquitin-like modifier-2/3.
    Figure Legend Snippet: Neuronal specificity and efficacy of RNA interference with SUMO2/3 and its effects on survival of mouse primary cortical neurons. At days in vitro (DIV) 3, primary cortical neurons were transduced with lentiviral particles expressing EGFP as a reporter and either SUMO2/3 or control microRNA driven by the neuron-specific synapsin promoter. ( A ) Verification of transduction efficacy and neuronal specificity of cultures transduced with lentiviral particles driven by the synapsin promoter. Immunohistochemistry was performed after paraformaldehyde (PFA) fixation on DIV 12 with antibodies against EGFP, microtubuli-associated protein 2 (MAP2), and nuclear counterstain with DAPI. The multiplicity of infection (MOI) was ∼50. Scale bar=100 μ m. ( B ) Verification of knockdown efficiency of SUMO2/3 versus control microRNAs (1=LacZ, 2=non-targeting scrambled) with and without OGD (45 minutes and 3 hours reoxygenation) shown by a representative western blot analysis. Neuronal cultures were analyzed on DIV 12 and subjected to SDS-PAGE. Membranes were probed with antibodies against SUMO2/3, EGFP, and GAPDH. EGFP expression corresponded to an equal MOI of lentiviral particles and concomitant microRNA expression. GAPDH served as a housekeeping protein and equal loading control. ( C ) SUMO2/3 microRNA does not influence baseline survival over time up to DIV 12. In brief, microscopic pictures of EGFP fluorescence (indicative for microRNA delivery) were taken at DIV 6, 9, and 12 as described in the ‘Materials and methods' section. EGFP-expressing neurons were counted and ratios were calculated for DIV 9/6 (indicated in blue) and DIV 12/9 (red) to evaluate cell survival over time. We assumed an effect size > 0.15 and performed a prospective power analysis with α =0.05 and β =0.20. There was no significant difference between groups in a two-way repeated-measures ANOVA followed by Tukey's post hoc analysis. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP2, microtubule-associated protein 2; OGD, oxygen–glucose deprivation; SUMO2/3, small ubiquitin-like modifier-2/3.

    Techniques Used: In Vitro, Transduction, Expressing, Immunohistochemistry, Infection, Western Blot, SDS Page, Fluorescence

    38) Product Images from "RhoA/C inhibits proliferation by inducing the synthesis of GPRC5A"

    Article Title: RhoA/C inhibits proliferation by inducing the synthesis of GPRC5A

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69481-2

    Knockout of GPRC5A in MDA-MB-231 cells does not inhibit proliferation. ( A ) Western Blot analysis of MDA-MB-231 wild-type and knockout cells. GAPDH was used as loading control (representative of n = 3). ( B ) Quantification of A. GPRC5A expression was normalized to GAPDH. Data of three independent experiments were analyzed using t-test. ****p
    Figure Legend Snippet: Knockout of GPRC5A in MDA-MB-231 cells does not inhibit proliferation. ( A ) Western Blot analysis of MDA-MB-231 wild-type and knockout cells. GAPDH was used as loading control (representative of n = 3). ( B ) Quantification of A. GPRC5A expression was normalized to GAPDH. Data of three independent experiments were analyzed using t-test. ****p

    Techniques Used: Knock-Out, Multiple Displacement Amplification, Western Blot, Expressing

    RhoA and RhoC induce the expression of GPRC5A. ( A ) GPRC5A mRNA level was measured by qRT-PCR following expression of RhoA, RhoC or GFP in MCF-10Atet cells (+ Dox) for 24 h. S29 was used as a housekeeping gene control. RNA levels after expression induction (+ Dox) were normalized to the untreated control (− Dox). Data of five independent experiments were quantified and analyzed using two-way ANOVA. ( B ) Representative Western Blot showing GPRC5A and EGFR protein levels following stimulation of RhoA, RhoC and GFP expressing MCF-10Atet cells for 0, 24 or 48 h with doxycycline (Dox). GAPDH and tubulin served as loading controls, respectively. Note that high expression of RhoC leads to decreased expression of RhoA, which has been described earlier 8 . ( C ) Quantification of B. GPRC5A protein level was normalized to GAPDH (left), EGFR protein level was normalized to tubulin (right). Data of three independent experiments were quantified and analyzed using two-way ANOVA. ( D ) GPRC5A mRNA level was measured by qRT-PCR after intoxication of MCF10A wild-type cells for 24 h with CNF1, CNF1 C866S, CNFY or CNFY C865S, respectively. S29 was used as a housekeeping gene control. RNA levels were normalized to the untreated (utr) control. Data of five independent experiments were quantified and analyzed using one-way-ANOVA. ( E ) Representative Western Blot (n = 3) showing GPRC5A and EGFR protein levels after intoxication with CNF1, CNF1 C866S, CNFY or CNFY C865S for 0, 24 or 48 h. GAPDH and tubulin served as loading controls. ( F ) Quantification of E. GPRC5A protein level was normalized to GAPDH (top), EGFR protein level was normalized to tubulin (bottom). The treatment with CNF1 and CNFY for two days GPRC5A expression was increased but the EGFR level was not affected. Data of three independent experiments were quantified and analyzed using one-way ANOVA. *p
    Figure Legend Snippet: RhoA and RhoC induce the expression of GPRC5A. ( A ) GPRC5A mRNA level was measured by qRT-PCR following expression of RhoA, RhoC or GFP in MCF-10Atet cells (+ Dox) for 24 h. S29 was used as a housekeeping gene control. RNA levels after expression induction (+ Dox) were normalized to the untreated control (− Dox). Data of five independent experiments were quantified and analyzed using two-way ANOVA. ( B ) Representative Western Blot showing GPRC5A and EGFR protein levels following stimulation of RhoA, RhoC and GFP expressing MCF-10Atet cells for 0, 24 or 48 h with doxycycline (Dox). GAPDH and tubulin served as loading controls, respectively. Note that high expression of RhoC leads to decreased expression of RhoA, which has been described earlier 8 . ( C ) Quantification of B. GPRC5A protein level was normalized to GAPDH (left), EGFR protein level was normalized to tubulin (right). Data of three independent experiments were quantified and analyzed using two-way ANOVA. ( D ) GPRC5A mRNA level was measured by qRT-PCR after intoxication of MCF10A wild-type cells for 24 h with CNF1, CNF1 C866S, CNFY or CNFY C865S, respectively. S29 was used as a housekeeping gene control. RNA levels were normalized to the untreated (utr) control. Data of five independent experiments were quantified and analyzed using one-way-ANOVA. ( E ) Representative Western Blot (n = 3) showing GPRC5A and EGFR protein levels after intoxication with CNF1, CNF1 C866S, CNFY or CNFY C865S for 0, 24 or 48 h. GAPDH and tubulin served as loading controls. ( F ) Quantification of E. GPRC5A protein level was normalized to GAPDH (top), EGFR protein level was normalized to tubulin (bottom). The treatment with CNF1 and CNFY for two days GPRC5A expression was increased but the EGFR level was not affected. Data of three independent experiments were quantified and analyzed using one-way ANOVA. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    Expression of GPRC5A reduces EGF stimulated phosphorylation of EGFR. ( A ) Western Blot analysis of EGFR phosphorylation following transient expression of GPRC5A. In MCF10A cells, RAI3 expression was induced by viral transduction using pMiBerry-GPRC5A or empty vector as control. After serum starvation for 48 h, the cells were stimulated with 0, 1.3 or 20 ng/ml EGF for five min. Representative Western Blots of three independent experiments are shown. GAPDH was used as loading control. ( B ) Quantification of A. Data of three independent experiments were quantified and analyzed using two-way ANOVA. ***p
    Figure Legend Snippet: Expression of GPRC5A reduces EGF stimulated phosphorylation of EGFR. ( A ) Western Blot analysis of EGFR phosphorylation following transient expression of GPRC5A. In MCF10A cells, RAI3 expression was induced by viral transduction using pMiBerry-GPRC5A or empty vector as control. After serum starvation for 48 h, the cells were stimulated with 0, 1.3 or 20 ng/ml EGF for five min. Representative Western Blots of three independent experiments are shown. GAPDH was used as loading control. ( B ) Quantification of A. Data of three independent experiments were quantified and analyzed using two-way ANOVA. ***p

    Techniques Used: Expressing, Western Blot, Transduction, Plasmid Preparation

    Knockout of GPRC5A in MCF10A cells inhibits EGF-dependent proliferation due to downregulation of EGFR. ( A ) Western Blot analysis of MCF10A wild-type and knockout cells in full medium and under serum starvation, respectively. GAPDH and tubulin were used as a loading controls. Representative Western Blot of three independent experiments are shown. ( B ) Quantification of A. GPRC5A expression was normalized to GAPDH, EGFR expression to tubulin. Data of three independent experiments were analyzed using one-way ANOVA. **p
    Figure Legend Snippet: Knockout of GPRC5A in MCF10A cells inhibits EGF-dependent proliferation due to downregulation of EGFR. ( A ) Western Blot analysis of MCF10A wild-type and knockout cells in full medium and under serum starvation, respectively. GAPDH and tubulin were used as a loading controls. Representative Western Blot of three independent experiments are shown. ( B ) Quantification of A. GPRC5A expression was normalized to GAPDH, EGFR expression to tubulin. Data of three independent experiments were analyzed using one-way ANOVA. **p

    Techniques Used: Knock-Out, Western Blot, Expressing

    39) Product Images from "In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1? Axis"

    Article Title: In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1? Axis

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2011.04.011

    Effects of PGC-1α Overexpression in Surf1 −/− Mice (A) Expression analysis of PGC-1α . (Left panel) The PGC-1α transcript from muscle of each mouse genotype (three animals/genotype) was retrotranscribed into cDNA, normalized to that of the Hprt gene, and expressed as time-fold variations relative to the values obtained from wild-type (WT) animals. Solid blue, WT; blue outline, PGC-1α : MCK-PGC-1α transgenic mouse; solid red, Surf1 −/− , constitutive Surf1 knockout mouse; red outline, Surf1 −/− /PGC-1α : Surf1 −/− /MCK-PGC-1α double mutant mouse. Error bars represent the standard deviation (SD). (Right panel) Western blot immunovisualization of skeletal muscle proteins of the different genotypes, listed as above. Densitometric analysis of each band, normalized against that of GAPDH, revealed the following variations, relative to WT. (1) PGC-1α, 4.3 in MCK-PGC-1α ; 0.5 in Surf1 −/− ; 2.2 in Surf1 −/− /PGC-1α . (2) COX1, 1.8 in MCK-PGC-1α ; 0.2 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (3) COX5a, 5.0 in MCK-PGC-1α ; ∼0.0 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (B) MtDNA analysis in different genotypes (three animals/genotype). Color code is as in (A). MtDNA is expressed as number of DNA molecules per cell ( Cree et al., 2008 ). Error bars represent SD. (C) MRC activities in the different genotypes (three animals/genotype), expressed as nmoles/min/mg of protein. Note that the activity of cII has been multiplied by 10 for visualization clarity. Color code is as in (A). CS, citrate synthase; CI-IV, MRC complexes I–IV. Error bars represent the standard deviation (SD). The asterisks represent the significance levels calculated by unpaired, Student's two-tailed t test: ∗ p
    Figure Legend Snippet: Effects of PGC-1α Overexpression in Surf1 −/− Mice (A) Expression analysis of PGC-1α . (Left panel) The PGC-1α transcript from muscle of each mouse genotype (three animals/genotype) was retrotranscribed into cDNA, normalized to that of the Hprt gene, and expressed as time-fold variations relative to the values obtained from wild-type (WT) animals. Solid blue, WT; blue outline, PGC-1α : MCK-PGC-1α transgenic mouse; solid red, Surf1 −/− , constitutive Surf1 knockout mouse; red outline, Surf1 −/− /PGC-1α : Surf1 −/− /MCK-PGC-1α double mutant mouse. Error bars represent the standard deviation (SD). (Right panel) Western blot immunovisualization of skeletal muscle proteins of the different genotypes, listed as above. Densitometric analysis of each band, normalized against that of GAPDH, revealed the following variations, relative to WT. (1) PGC-1α, 4.3 in MCK-PGC-1α ; 0.5 in Surf1 −/− ; 2.2 in Surf1 −/− /PGC-1α . (2) COX1, 1.8 in MCK-PGC-1α ; 0.2 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (3) COX5a, 5.0 in MCK-PGC-1α ; ∼0.0 in Surf1 −/− ; 1.0 in Surf1 −/− /PGC-1α . (B) MtDNA analysis in different genotypes (three animals/genotype). Color code is as in (A). MtDNA is expressed as number of DNA molecules per cell ( Cree et al., 2008 ). Error bars represent SD. (C) MRC activities in the different genotypes (three animals/genotype), expressed as nmoles/min/mg of protein. Note that the activity of cII has been multiplied by 10 for visualization clarity. Color code is as in (A). CS, citrate synthase; CI-IV, MRC complexes I–IV. Error bars represent the standard deviation (SD). The asterisks represent the significance levels calculated by unpaired, Student's two-tailed t test: ∗ p

    Techniques Used: Pyrolysis Gas Chromatography, Over Expression, Mouse Assay, Expressing, Transgenic Assay, Knock-Out, Mutagenesis, Standard Deviation, Western Blot, Activity Assay, Two Tailed Test

    40) Product Images from "STAT3 Localizes in Mitochondria-Associated ER Membranes Instead of in Mitochondria"

    Article Title: STAT3 Localizes in Mitochondria-Associated ER Membranes Instead of in Mitochondria

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.00274

    Re-examining the existence and function of ‘mitochondrial STAT3.’ (A) Purification of mitochondria by sonication. (B) Purification of mitochondria by trypsinization. (C) Purification of mitochondria by washing with high concentration of salt combined with trypsinization. GRP78 was used as the ER marker; ATP5A, NDUFA9, NDUFA13, and VDAC were used as the mitochondrial marker; GAPDH was used as the cytosolic marker. (D) Sucrose density centrifuge of digitonin-solubilized mitochondria followed by Western blot analysis of STAT3 and mitochondrial complexes protein. (E) Co-immunoprecipitation experiment in digitonin-solubilized crude mitochondria. (F) ChIP-qPCR detection of STAT3-binding on mitochondrial DNA in mouse embryonic stem cells. (G) Serum reintroduction experiment in Neuro2A cells. (H) Quantification of Western blot results (G) in three independent experiments. * p
    Figure Legend Snippet: Re-examining the existence and function of ‘mitochondrial STAT3.’ (A) Purification of mitochondria by sonication. (B) Purification of mitochondria by trypsinization. (C) Purification of mitochondria by washing with high concentration of salt combined with trypsinization. GRP78 was used as the ER marker; ATP5A, NDUFA9, NDUFA13, and VDAC were used as the mitochondrial marker; GAPDH was used as the cytosolic marker. (D) Sucrose density centrifuge of digitonin-solubilized mitochondria followed by Western blot analysis of STAT3 and mitochondrial complexes protein. (E) Co-immunoprecipitation experiment in digitonin-solubilized crude mitochondria. (F) ChIP-qPCR detection of STAT3-binding on mitochondrial DNA in mouse embryonic stem cells. (G) Serum reintroduction experiment in Neuro2A cells. (H) Quantification of Western blot results (G) in three independent experiments. * p

    Techniques Used: Purification, Sonication, Concentration Assay, Marker, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

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    Immunoprecipitation:

    Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons
    Article Snippet: .. The following commercially available antibodies were used for immunoblot analysis and immunoprecipitation: mouse monoclonal anti-FLAG-peroxidase (M2; 1:3,000, Sigma, #A8592); anti-hemagglutinin (HA)-peroxidase (3F10; 1:1,000; Roche, #12013819001); anti-V5-peroxidase (1:5,000; Invitrogen, #R961-25); anti-GAPDH antibody (6C5; 1:4,000; Millipore, #MAB374); anti-GCN5 (C26A10; 1:1,000; Cell Signaling Technology, #3305); and rabbit polyclonal anti-acetylated-lysine (1:1,000; Cell Signaling Technology, #9441). .. All other antibodies were purchased from Santa Cruz Biotechnology, including rabbit polyclonal anti-CDK5 (C8; 1:1,000; #sc-173), anti-His probe (H15; 1:1,000; #sc-803), anti-SIRT1 (H-300; 1:1,000; #sc-15404), anti-SIRT2 (H-95; 1:1,000; #sc-20966), anti-p35 (C19; 1:1,000; #sc-820), mouse monoclonal anti-CDK5 (J3; 1:1,000; #sc-6247), anti-CHIP (G2; 1:1,000), and goat polyclonal anti-SOD-1 (C17; 1:1,000; #sc-8637) and anti-Lamin A/C (N18; 1:1,000; #sc-6215).

    Incubation:

    Article Title: Gas6 derived from cancer-associated fibroblasts promotes migration of Axl-expressing lung cancer cells during chemotherapy
    Article Snippet: .. Proteins were then transferred onto nylon membranes (Amersham, Buckinghamshire, UK) and incubated with mouse anti-α-SMA antibody (clone 1A4, Sigma-Aldrich), mouse anti-vimentin antibody (vim3B4, Dako), rabbit anti-PDGFR-α antibody (#3164, Cell Signaling Technology), rabbit anti-PDGFR-β antibody (#3169, Cell Signaling Technology), goat anti-Axl antibody (AF154, R & D Systems), rabbit anti-phospho Axl antibody (AF2228, R & D Systems), goat anti-Gas6 antibody (AF885, R & D Systems) or mouse anti-GAPDH antibody (Chemicon, Temecula, CA, USA) according to the manufacturers’ protocols. .. Proteins were detected with horseradish peroxidase-conjugated anti-rabbit, anti-goat, or anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and ECL reagents (GE Healthcare, Milwaukee, WI, USA).

    Article Title: Individual differences in the expression of conditioned fear are associated with endogenous fibroblast growth factor 2
    Article Snippet: .. Proteins were transferred to nitrocellulose membranes, and nonspecific immunoreactivity was blocked with 5% nonfat dry milk/3% BSA in TBST for 60 min. Membranes were incubated overnight at 4°C with anti-FGF2 antibody (1:500; Abcam) or anti-GAPDH antibody (1:500; Millipore). .. After incubation in secondary antibody (HRP-conjugated goat anti-rabbit/anti-mouse IgG; BioRad) visualization was conducted using the ECL detection method in an ImageQuant LAS 500.

    Article Title: Downregulation of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in a co-culture system with human stimulated X-linked CGD neutrophils
    Article Snippet: .. For immunodetection, membranes were blocked in 5% BSA in TBS with 0.1% Tween-20 (TBST) and incubated in TBST with eNOS and phosphorylated-eNOS(Ser1177) antibodies (Cell life signaling, Danvers, MA, USA) (1:100 dilution), anti-EDN1 antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:500), anti- NFκB (p65) antibody, and anti-phosphorylated NFκB (p65) at Ser536 position (St. John’s Laboratory, London, UK) (1:500), and anti-GAPDH antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:1000) with chemiluminescent detection. .. Blots were incubated with peroxidase-labeled anti-rabbit or anti-mouse IgG secondary antibody (1:20,000; cat# PI-1000 (Rabbit), PI-2000 (mouse), Vector Labs, Burlingame, CA, USA) at room temperature, followed by enhanced chemiluminescence detection (Immobilon Western HRP substrate Luminol Reagent, Millipore, Billerica, MA, USA).

    Article Title: Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma
    Article Snippet: .. After blocking with a 10% solution of defatted milk, the membranes were incubated overnight at 4°C with the following primary antibodies : anti-Kv1.3 (1:200, rabbit polyclonal, Alamone Labs APC-101); anti-GAPDH (1:1000, mouse monoclonal, Millipore MAB374). .. After washing, the membranes were developed using corresponding anti-mouse or anti-rabbit secondary antibodies (Calbiochem).

    other:

    Article Title: cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1
    Article Snippet: Antibodies used were anti-CCN1 (R & D Systems, #AF4055), anti-GAPDH (Millipore, #MAB374), anti-MKL1 (Santa Cruz #sc21558), anti-BrdU (Sigma; #B253).

    Immunodetection:

    Article Title: Downregulation of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in a co-culture system with human stimulated X-linked CGD neutrophils
    Article Snippet: .. For immunodetection, membranes were blocked in 5% BSA in TBS with 0.1% Tween-20 (TBST) and incubated in TBST with eNOS and phosphorylated-eNOS(Ser1177) antibodies (Cell life signaling, Danvers, MA, USA) (1:100 dilution), anti-EDN1 antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:500), anti- NFκB (p65) antibody, and anti-phosphorylated NFκB (p65) at Ser536 position (St. John’s Laboratory, London, UK) (1:500), and anti-GAPDH antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:1000) with chemiluminescent detection. .. Blots were incubated with peroxidase-labeled anti-rabbit or anti-mouse IgG secondary antibody (1:20,000; cat# PI-1000 (Rabbit), PI-2000 (mouse), Vector Labs, Burlingame, CA, USA) at room temperature, followed by enhanced chemiluminescence detection (Immobilon Western HRP substrate Luminol Reagent, Millipore, Billerica, MA, USA).

    Blocking Assay:

    Article Title: Tumor-reducing effect of the clinically used drug clofazimine in a SCID mouse model of pancreatic ductal adenocarcinoma
    Article Snippet: .. After blocking with a 10% solution of defatted milk, the membranes were incubated overnight at 4°C with the following primary antibodies : anti-Kv1.3 (1:200, rabbit polyclonal, Alamone Labs APC-101); anti-GAPDH (1:1000, mouse monoclonal, Millipore MAB374). .. After washing, the membranes were developed using corresponding anti-mouse or anti-rabbit secondary antibodies (Calbiochem).

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    <t>DAP5</t> positively regulates cap-independent translation of DSCR1.4 by binding to 5′UTR of DSCR1.4. a , b DAP5 interacts with hDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated hDSCR1.4 5′UTR constructs were incubated with SHSY5Y cell extracts. The region interacting with DAP5 was confirmed by Western blot. <t>GAPDH</t> and hDSCR1.4 were used as negative control. Nonbiotinylated hDSCR1.4 UTR was used as competitor. c DAP5 binds to proximal 136 nucleotides of mDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated mDSCR1.4 5′UTR transcripts were incubated with N2A cell extracts. Binding region was verified by Western blot. d , e A reduction of DAP5 decreases cap-independent translation efficiency of DSCR1.4 5′UTR. d SHSY5Y and e N2A cells were transfected with control siRNA (siCon) or DAP5 siRNA (siDAP5) and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and siCon transfected cells was set as 1. The bars represent the mean ± SEM ( n = 7, n = 7). f , g DAP5 overexpression upregulated cap-independent translation of DSCR1.4 5′UTR. f SHSY5Y and g N2A cells were transfected with Flag Mock or Flag DAP5 plasmids and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and Flag Mock transfected cells was set as 1. The bars represent the mean ± SEM ( n = 5, n = 5). h , i DAP5 deficiency results in reduction of DSCR1.4 protein levels. siCon or siDAP5 was transfected on h SHSY5Y and i N2A cells. DAP5 knockdown was confirmed by Western blot using anti-DAP5. j , k Overexpressed DAP5 increases DSCR1.4 protein levels. j SHSY5Y and k N2A cells were transfected with Flag Mock or Flag DAP5 and incubated for 24 h. DAP5 overexpression was verified by Western blot using anti-Flag antibody. l The increase of DSCR1.4 expressions by DAP5 overexpression results from cap-independent translation. SHSY5Ycells were transfected with Flag Mock or Flag DAP5 and 18 h later incubated and followed by 200 μM rapamycin treatment for 6 h. The rapamycin activity was analyzed by phosphorylation status of S6RP protein. DAP5 overexpression was confirmed by anti-Flag antibody. Data information: In d – g , * P
    Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    DAP5 positively regulates cap-independent translation of DSCR1.4 by binding to 5′UTR of DSCR1.4. a , b DAP5 interacts with hDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated hDSCR1.4 5′UTR constructs were incubated with SHSY5Y cell extracts. The region interacting with DAP5 was confirmed by Western blot. GAPDH and hDSCR1.4 were used as negative control. Nonbiotinylated hDSCR1.4 UTR was used as competitor. c DAP5 binds to proximal 136 nucleotides of mDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated mDSCR1.4 5′UTR transcripts were incubated with N2A cell extracts. Binding region was verified by Western blot. d , e A reduction of DAP5 decreases cap-independent translation efficiency of DSCR1.4 5′UTR. d SHSY5Y and e N2A cells were transfected with control siRNA (siCon) or DAP5 siRNA (siDAP5) and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and siCon transfected cells was set as 1. The bars represent the mean ± SEM ( n = 7, n = 7). f , g DAP5 overexpression upregulated cap-independent translation of DSCR1.4 5′UTR. f SHSY5Y and g N2A cells were transfected with Flag Mock or Flag DAP5 plasmids and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and Flag Mock transfected cells was set as 1. The bars represent the mean ± SEM ( n = 5, n = 5). h , i DAP5 deficiency results in reduction of DSCR1.4 protein levels. siCon or siDAP5 was transfected on h SHSY5Y and i N2A cells. DAP5 knockdown was confirmed by Western blot using anti-DAP5. j , k Overexpressed DAP5 increases DSCR1.4 protein levels. j SHSY5Y and k N2A cells were transfected with Flag Mock or Flag DAP5 and incubated for 24 h. DAP5 overexpression was verified by Western blot using anti-Flag antibody. l The increase of DSCR1.4 expressions by DAP5 overexpression results from cap-independent translation. SHSY5Ycells were transfected with Flag Mock or Flag DAP5 and 18 h later incubated and followed by 200 μM rapamycin treatment for 6 h. The rapamycin activity was analyzed by phosphorylation status of S6RP protein. DAP5 overexpression was confirmed by anti-Flag antibody. Data information: In d – g , * P

    Journal: Cell Death & Disease

    Article Title: DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA

    doi: 10.1038/s41419-018-1299-x

    Figure Lengend Snippet: DAP5 positively regulates cap-independent translation of DSCR1.4 by binding to 5′UTR of DSCR1.4. a , b DAP5 interacts with hDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated hDSCR1.4 5′UTR constructs were incubated with SHSY5Y cell extracts. The region interacting with DAP5 was confirmed by Western blot. GAPDH and hDSCR1.4 were used as negative control. Nonbiotinylated hDSCR1.4 UTR was used as competitor. c DAP5 binds to proximal 136 nucleotides of mDSCR1.4 5′UTR. In vitro transcribed biotin-conjugated mDSCR1.4 5′UTR transcripts were incubated with N2A cell extracts. Binding region was verified by Western blot. d , e A reduction of DAP5 decreases cap-independent translation efficiency of DSCR1.4 5′UTR. d SHSY5Y and e N2A cells were transfected with control siRNA (siCon) or DAP5 siRNA (siDAP5) and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and siCon transfected cells was set as 1. The bars represent the mean ± SEM ( n = 7, n = 7). f , g DAP5 overexpression upregulated cap-independent translation of DSCR1.4 5′UTR. f SHSY5Y and g N2A cells were transfected with Flag Mock or Flag DAP5 plasmids and 24 h later with pRF bicistronic vectors. Luciferase activity of pRF mock and Flag Mock transfected cells was set as 1. The bars represent the mean ± SEM ( n = 5, n = 5). h , i DAP5 deficiency results in reduction of DSCR1.4 protein levels. siCon or siDAP5 was transfected on h SHSY5Y and i N2A cells. DAP5 knockdown was confirmed by Western blot using anti-DAP5. j , k Overexpressed DAP5 increases DSCR1.4 protein levels. j SHSY5Y and k N2A cells were transfected with Flag Mock or Flag DAP5 and incubated for 24 h. DAP5 overexpression was verified by Western blot using anti-Flag antibody. l The increase of DSCR1.4 expressions by DAP5 overexpression results from cap-independent translation. SHSY5Ycells were transfected with Flag Mock or Flag DAP5 and 18 h later incubated and followed by 200 μM rapamycin treatment for 6 h. The rapamycin activity was analyzed by phosphorylation status of S6RP protein. DAP5 overexpression was confirmed by anti-Flag antibody. Data information: In d – g , * P

    Article Snippet: Proteins were detected by monoclonal anti-FLUC(Abcam), anti-DAP5 (Abcam), polyclonal anti-DSCR1 (Sigma-Aldrich), anti-GAPDH (Millipore), anti-Flag (Cell signaling), anti-Lamin B (Santa Cruz), and Horseradish peroxidase (HRP)-conjugated mouse (Thermo Scientific) and rabbit (Promega) secondary antibodies.

    Techniques: Binding Assay, In Vitro, Construct, Incubation, Western Blot, Negative Control, Transfection, Luciferase, Activity Assay, Over Expression

    BDNF makes cap-independent translation of DSCR1.4 mRNA more actively by increasing DAP5 expression. a , b BDNF treatment on DIV 3 hippocampal neuron increases protein levels of DAP5 and DSCR1.4 but not DSCR1.4 mRNA level. Vehicle (DDW) or 30 μM BDNF were treated for 1 h. a The protein levels were confirmed by Western blot. GAPDH and phosphorylation of ERK were used as a loading control and marker of BDNF activity, respectively. b Endogenous DSCR1.4 mRNA levels were analyzed by qRT-PCR and were normalized to β-actin. The bars represent the mean ± SEM ( n = 3). c Cap-independent translation is essential for DSCR1.4 protein accumulation by BDNF. DIV 3 hippocampal neurons were treated with vehicle (DMSO), 100 μM RAD001(RAD) or 50 mg/ml cycloheximide (CHX) for 3 h followed by BDNF treatment for 1 h. The levels of each protein were confirmed by Western blot. The numbers at the bottom indicate the fold relative to a vehicle. The amount of DSCR1.4 was normalized to GAPDH. d , e BDNF raises cap-independent translation activity of DSCR1.4 mRNA. At 24 h after d pRF hDSCR1.4 5′UTR or e pRF mDSCR1.4 5′UTR vectors were transfected into DIV 2 hippocampal neurons, Vehicle (DDW) and BDNF were treated to the neurons for 1 h. The bars represent the mean ± SEM ( e ; n = 5, F; n = 3). f BDNF increases the interaction between DAP5 and DSCR1.4 5′UTR. In vitro transcribed biotin-DSCR1.4 5′UTR was incubated with extracts of the vehicle (DDW) or 30 μM BDNF-treated DIV 3 mouse hippocampal neurons. DAP5 binding was measured by Western blot. Phospho-ERK was used to confirm the activity of BDNF. GADPH was used as a loading control and negative control. g , h BDNF increases the cap-independent local translation of DSCR1.4 mRNA in axon as well as soma. EGFP and pRF mDSCR1.4 5′ 3′ UTR vectors were co-transfected into DIV 2 mouse hippocampal neurons. At 24 h later, 100 μM anisomycin was treated for 3 h and then 30 μM BDNF was treated for 1 h, followed by 5 μM puromycin treatment for 40 min. To detect newly synthesized FLUC proteins, Puro-PLA assay was conducted. g Representative image obtained from confocal microscopy. h The graph shows relative fluorescence intensity measured by Image J. The bars represent the mean ± SEM (Vehicle; n = 11, Anisomycin; n = 12, BDNF; n = 11, BDNF + Anisomycin; n = 11). Scale bar, 30 μm. Data information: In d , e , h , * P

    Journal: Cell Death & Disease

    Article Title: DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA

    doi: 10.1038/s41419-018-1299-x

    Figure Lengend Snippet: BDNF makes cap-independent translation of DSCR1.4 mRNA more actively by increasing DAP5 expression. a , b BDNF treatment on DIV 3 hippocampal neuron increases protein levels of DAP5 and DSCR1.4 but not DSCR1.4 mRNA level. Vehicle (DDW) or 30 μM BDNF were treated for 1 h. a The protein levels were confirmed by Western blot. GAPDH and phosphorylation of ERK were used as a loading control and marker of BDNF activity, respectively. b Endogenous DSCR1.4 mRNA levels were analyzed by qRT-PCR and were normalized to β-actin. The bars represent the mean ± SEM ( n = 3). c Cap-independent translation is essential for DSCR1.4 protein accumulation by BDNF. DIV 3 hippocampal neurons were treated with vehicle (DMSO), 100 μM RAD001(RAD) or 50 mg/ml cycloheximide (CHX) for 3 h followed by BDNF treatment for 1 h. The levels of each protein were confirmed by Western blot. The numbers at the bottom indicate the fold relative to a vehicle. The amount of DSCR1.4 was normalized to GAPDH. d , e BDNF raises cap-independent translation activity of DSCR1.4 mRNA. At 24 h after d pRF hDSCR1.4 5′UTR or e pRF mDSCR1.4 5′UTR vectors were transfected into DIV 2 hippocampal neurons, Vehicle (DDW) and BDNF were treated to the neurons for 1 h. The bars represent the mean ± SEM ( e ; n = 5, F; n = 3). f BDNF increases the interaction between DAP5 and DSCR1.4 5′UTR. In vitro transcribed biotin-DSCR1.4 5′UTR was incubated with extracts of the vehicle (DDW) or 30 μM BDNF-treated DIV 3 mouse hippocampal neurons. DAP5 binding was measured by Western blot. Phospho-ERK was used to confirm the activity of BDNF. GADPH was used as a loading control and negative control. g , h BDNF increases the cap-independent local translation of DSCR1.4 mRNA in axon as well as soma. EGFP and pRF mDSCR1.4 5′ 3′ UTR vectors were co-transfected into DIV 2 mouse hippocampal neurons. At 24 h later, 100 μM anisomycin was treated for 3 h and then 30 μM BDNF was treated for 1 h, followed by 5 μM puromycin treatment for 40 min. To detect newly synthesized FLUC proteins, Puro-PLA assay was conducted. g Representative image obtained from confocal microscopy. h The graph shows relative fluorescence intensity measured by Image J. The bars represent the mean ± SEM (Vehicle; n = 11, Anisomycin; n = 12, BDNF; n = 11, BDNF + Anisomycin; n = 11). Scale bar, 30 μm. Data information: In d , e , h , * P

    Article Snippet: Proteins were detected by monoclonal anti-FLUC(Abcam), anti-DAP5 (Abcam), polyclonal anti-DSCR1 (Sigma-Aldrich), anti-GAPDH (Millipore), anti-Flag (Cell signaling), anti-Lamin B (Santa Cruz), and Horseradish peroxidase (HRP)-conjugated mouse (Thermo Scientific) and rabbit (Promega) secondary antibodies.

    Techniques: Expressing, Western Blot, Marker, Activity Assay, Quantitative RT-PCR, Transfection, In Vitro, Incubation, Binding Assay, Negative Control, Synthesized, Proximity Ligation Assay, Confocal Microscopy, Fluorescence

    hDSCR1.4 and mDSCR1.4 mRNA are cap-independently translated and have cis-regulatory elements in their 5′UTRs. a Cap-independent translational regulation contributes to DSCR1.4 protein expression. SHSY5Y cells were treated with DMSO or 200 μM rapamycin or 50 mg/ml cycloheximide for the indicated times. The levels of endogenous proteins were measured by Western blotting (WB) using anti-DSCR1.4, anti-phosphoS6RP, anti-GAPDH antibodies. GAPDH was used as a loading control. The activity of rapamycin was analyzed by the phosphorylation status of S6RP. The numbers at the bottom indicate the fold increases relative to control. The amount of DSCR1.4 was normalized to GAPDH. b Schematic representation of pRF bicistronic luciferase plasmids used for observing cap-independent translation activity of human and mouse DSCR1.4 5′UTR. c , d hDSCR1.4 5′UTR and mDSCR1.4 5′UTR induce cap-independent translation initiation. c SHSY5Y and d N2A cells were transfected with the bicistronic reporter plasmids and were incubated for 24 h. pRF β-globin was used as a negative control. pRF EMCV and pRF p53 were used as the positive control. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 4). e , f hDSCR1.4 5′UTR and mDSCR1.4 5′UTR prefer cap-independent translation to cap-dependent translation. e SHSY5Y cells were transfected with in vitro transcribed m7G capped or ApppG capped hDSCR1.4 5′UTR-FLUC. f N2A cells were transfected with in vitro transcribed m7G capped or ApppG capped mDSCR1.4 5′UTR-FLUC. Transfected cells were incubated for 6 h and were harvested. Translation activity is shown as the ratio of FLUC to FLUC mRNA. Translation activity of m7G capped transcripts was set as 1. The bars represent the mean ± SEM ( n = 4, n = 3). g , i The 5′ proximal 119 nucleotides sequence of hDSCR1.4 5′UTR is important for cap-independent translation activity of hDSCR1.4 5′UTR. pRF plasmids with serial deletion constructs and six nucleotides mutant construct were transfected into g SHSY5Y cells and i mouse primary hippocampal neurons. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3). h , j The 5′ proximal 136 nucleotides are essential for cap-independent translation activity of mDSCR1.4 5′UTR. Indicated pRF plasmids were transfected into h N2A cells and j mouse hippocampal neurons. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 3). Data information: In c – j , * P

    Journal: Cell Death & Disease

    Article Title: DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA

    doi: 10.1038/s41419-018-1299-x

    Figure Lengend Snippet: hDSCR1.4 and mDSCR1.4 mRNA are cap-independently translated and have cis-regulatory elements in their 5′UTRs. a Cap-independent translational regulation contributes to DSCR1.4 protein expression. SHSY5Y cells were treated with DMSO or 200 μM rapamycin or 50 mg/ml cycloheximide for the indicated times. The levels of endogenous proteins were measured by Western blotting (WB) using anti-DSCR1.4, anti-phosphoS6RP, anti-GAPDH antibodies. GAPDH was used as a loading control. The activity of rapamycin was analyzed by the phosphorylation status of S6RP. The numbers at the bottom indicate the fold increases relative to control. The amount of DSCR1.4 was normalized to GAPDH. b Schematic representation of pRF bicistronic luciferase plasmids used for observing cap-independent translation activity of human and mouse DSCR1.4 5′UTR. c , d hDSCR1.4 5′UTR and mDSCR1.4 5′UTR induce cap-independent translation initiation. c SHSY5Y and d N2A cells were transfected with the bicistronic reporter plasmids and were incubated for 24 h. pRF β-globin was used as a negative control. pRF EMCV and pRF p53 were used as the positive control. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 4). e , f hDSCR1.4 5′UTR and mDSCR1.4 5′UTR prefer cap-independent translation to cap-dependent translation. e SHSY5Y cells were transfected with in vitro transcribed m7G capped or ApppG capped hDSCR1.4 5′UTR-FLUC. f N2A cells were transfected with in vitro transcribed m7G capped or ApppG capped mDSCR1.4 5′UTR-FLUC. Transfected cells were incubated for 6 h and were harvested. Translation activity is shown as the ratio of FLUC to FLUC mRNA. Translation activity of m7G capped transcripts was set as 1. The bars represent the mean ± SEM ( n = 4, n = 3). g , i The 5′ proximal 119 nucleotides sequence of hDSCR1.4 5′UTR is important for cap-independent translation activity of hDSCR1.4 5′UTR. pRF plasmids with serial deletion constructs and six nucleotides mutant construct were transfected into g SHSY5Y cells and i mouse primary hippocampal neurons. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3). h , j The 5′ proximal 136 nucleotides are essential for cap-independent translation activity of mDSCR1.4 5′UTR. Indicated pRF plasmids were transfected into h N2A cells and j mouse hippocampal neurons. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean ± SEM ( n = 3, n = 3). Data information: In c – j , * P

    Article Snippet: Proteins were detected by monoclonal anti-FLUC(Abcam), anti-DAP5 (Abcam), polyclonal anti-DSCR1 (Sigma-Aldrich), anti-GAPDH (Millipore), anti-Flag (Cell signaling), anti-Lamin B (Santa Cruz), and Horseradish peroxidase (HRP)-conjugated mouse (Thermo Scientific) and rabbit (Promega) secondary antibodies.

    Techniques: Expressing, Western Blot, Activity Assay, Luciferase, Transfection, Incubation, Negative Control, Positive Control, Plasmid Preparation, In Vitro, Sequencing, Construct, Mutagenesis

    TNF-α disrupted the autophagic flux in microglia. (A) Dose-dependent effect of the lysosome inhibitor BafA1 treatment for 2 h on LC3II accumulation in BV2 cells, as evaluated by western blotting. N = 3. (B) Effect of TNF-α on LC3II levels during lysosome inhibition. BV2 cells were treated with 5 ng/ml TNF-α for 24 h, and then added with BafA1(50 nM) for 2 h before subjected to western blotting. Actin served as loading controls in panels A,B . N = 4. (C,D) Autophagic assay in microglia treated with TNF-α, BafA1, or in combination. BV2 cells were transfected with RFP-GFP-tandem fluorescent LC3 cDNA for 24 h before treatment. Confocal microscope pictures showing yellow (GFP and RFP overlap) and red LC3 puncta formation in different groups. Scale bar, 10 μm. LC3 dots were visualized and quantified from at least 30 cells per group. (E) Effect of TNF-α on lysosomal biogenesis. BV2 cells were treated with 5 ng/ml TNF-α for 3, 12, or 24 h. The cytosolic and nuclear fractions were subjected to western blotting analysis of TFEB, with GAPDH and Histone 2B as the cytosolic and nuclear loading controls, respectively. (F,G) Effect of TNF-α on lysosomal protein LAMP1 (F) and LAMP2 (G) levels in BV2 cells. N = 3. ∗ P

    Journal: Frontiers in Aging Neuroscience

    Article Title: A Critical Role of Autophagy in Regulating Microglia Polarization in Neurodegeneration

    doi: 10.3389/fnagi.2018.00378

    Figure Lengend Snippet: TNF-α disrupted the autophagic flux in microglia. (A) Dose-dependent effect of the lysosome inhibitor BafA1 treatment for 2 h on LC3II accumulation in BV2 cells, as evaluated by western blotting. N = 3. (B) Effect of TNF-α on LC3II levels during lysosome inhibition. BV2 cells were treated with 5 ng/ml TNF-α for 24 h, and then added with BafA1(50 nM) for 2 h before subjected to western blotting. Actin served as loading controls in panels A,B . N = 4. (C,D) Autophagic assay in microglia treated with TNF-α, BafA1, or in combination. BV2 cells were transfected with RFP-GFP-tandem fluorescent LC3 cDNA for 24 h before treatment. Confocal microscope pictures showing yellow (GFP and RFP overlap) and red LC3 puncta formation in different groups. Scale bar, 10 μm. LC3 dots were visualized and quantified from at least 30 cells per group. (E) Effect of TNF-α on lysosomal biogenesis. BV2 cells were treated with 5 ng/ml TNF-α for 3, 12, or 24 h. The cytosolic and nuclear fractions were subjected to western blotting analysis of TFEB, with GAPDH and Histone 2B as the cytosolic and nuclear loading controls, respectively. (F,G) Effect of TNF-α on lysosomal protein LAMP1 (F) and LAMP2 (G) levels in BV2 cells. N = 3. ∗ P

    Article Snippet: The sources for primary antibodies were listed as follows: anti-β-actin, anti-p62, anti-lysosome-associated membrane protein 1 (LAMP1) and anti-β-tubulin from Sigma-Aldrich (St. Louis, MO, United States); anti-LC3, anti-histone 2B and anti-LAMP2 (Abcam, Cambridge, United Kingdom), anti-p-mammalian target of rapamycin (mTOR) [Cell Signaling Technology (CST), 5536s], anti-mTOR (CST, 2983s), anti-p-AKT (CST, 4060), anti-AKT (CST, 4691) and anti-cleaved caspase-3 (CST, 9664); anti-transcription factor EB (TFEB) (Proteintech, Chicago, IL, United States), and anti-GAPDH (Millipore, Billerica, MA, United States).

    Techniques: Western Blot, Inhibition, Transfection, Microscopy

    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

    Fig. 2 Effect of pyridone 6 on the activity of STAT3, MMPs, and TGF-β in human AAA tissue culture. Effect of pyridone 6 on STAT3 activity was evaluated by immunoblotting for activated (phosphorylated) STAT3 (P-STAT3) and total STAT3 after incubation of the AAA tissue with the indicated concentration of pyridone 6 for 48 h. ( A ) Representative images of immunoblotting for P-STAT3 and STAT3 are shown, and GAPDH was used as the internal control. ( B ) Expression levels of P-STAT3 normalized to that of GAPDH are represented. ( C ) Expression levels of STAT3 normalized to that of GAPDH are represented. ( D ) Secretions of MMP-9 and -2 were evaluated by gelatin zymography of the conditioned media. After collecting the basal secretion for 48 h, AAA tissue culture was treated with or without 100 ng/mL IL-6 and the indicated concentrations of pyridone 6. ( E ) Basal secretions of TGF-β family are shown in the conditioned media after 48 h of culture. ( F ) The effect of 10 µM pyridone 6 on the secretions of TGF-β family of cytokines is represented. Data are expressed as means±standard errors. The sample without pyridone 6 treatment was assigned a value of 1. * p

    Journal: Annals of Vascular Diseases

    Article Title: Cytokine Profile of Human Abdominal Aortic Aneurysm: Involvement of JAK/STAT Pathway

    doi: 10.3400/avd.oa.17-00086

    Figure Lengend Snippet: Fig. 2 Effect of pyridone 6 on the activity of STAT3, MMPs, and TGF-β in human AAA tissue culture. Effect of pyridone 6 on STAT3 activity was evaluated by immunoblotting for activated (phosphorylated) STAT3 (P-STAT3) and total STAT3 after incubation of the AAA tissue with the indicated concentration of pyridone 6 for 48 h. ( A ) Representative images of immunoblotting for P-STAT3 and STAT3 are shown, and GAPDH was used as the internal control. ( B ) Expression levels of P-STAT3 normalized to that of GAPDH are represented. ( C ) Expression levels of STAT3 normalized to that of GAPDH are represented. ( D ) Secretions of MMP-9 and -2 were evaluated by gelatin zymography of the conditioned media. After collecting the basal secretion for 48 h, AAA tissue culture was treated with or without 100 ng/mL IL-6 and the indicated concentrations of pyridone 6. ( E ) Basal secretions of TGF-β family are shown in the conditioned media after 48 h of culture. ( F ) The effect of 10 µM pyridone 6 on the secretions of TGF-β family of cytokines is represented. Data are expressed as means±standard errors. The sample without pyridone 6 treatment was assigned a value of 1. * p

    Article Snippet: Immunoblotting was performed for phosphorylated STAT3 (Tyr705, #9145, Cell Signaling Technology, Danvers, MA, USA), total STAT3 (#4904, Cell Signaling Technology), and GAPDH (#MAB374, Millipore), which served as the internal reference.

    Techniques: Activity Assay, Incubation, Concentration Assay, Expressing, Zymography