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Santa Cruz Biotechnology mouse anti actin
Interfering with SMN circRNAs biogenesis affects the expression of SMN protein. ( A ) Western blot analysis of DHX9 in DHX9-depleted cells (HEK293T). <t>Actin</t> was evaluated as loading control. ( B ) qPCR analysis for indicated back-splice junctions in DHX9-depleted cells (HEK293T). CircBRIP1, known to be regulated by DHX9 ( 29 ), was used as positive control. RPL34 was used to normalize <t>RNA</t> content in parallel RT-PCR reactions omitting RNAse R treatment (mean ± SD, n = 3; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; unpaired t-test). ( C ) Schematic representation of splicing and back-splicing events that generate the two exon 8-containing SMN circRNA, as indicated in the figure. Exons are represented as boxes and introns as lines. In blue and in white are indicated the coding and the non-coding exons, respectively. Shown below is the nucleotide sequence of SMN exon 8 and in red is indicated the cryptic 5′ splice site (c 5′SS) involved in the back-splicing event with the 3′ splice site of exon 6. The sequence of ASO-E8 is highlighted in the rectangle. pA stands for poly A site and 3′dwr stands for 3′ downstream region indicating a cryptic exon of 98 bp involved in circularization and located downstream from exon 8 of SMN . ( D ) Representative images of RT-PCR analysis for the indicated SMN2 circRNAs in ASO treated cells (GM03813). ASO-C is a control morpholino. ( E ) Representative images (left panel) and densitometry (right panel) of western blot for SMN and GEMIN 2 in ASO treated cells (GM03813). Actin was evaluated as loading control (mean ± SD, n = 5; * P ≤ 0.05, ns not significant; unpaired t -test).
Mouse Anti Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Sam68 binds Alu-rich introns in SMN and promotes pre-mRNA circularization"

Article Title: Sam68 binds Alu-rich introns in SMN and promotes pre-mRNA circularization

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz1117

Interfering with SMN circRNAs biogenesis affects the expression of SMN protein. ( A ) Western blot analysis of DHX9 in DHX9-depleted cells (HEK293T). Actin was evaluated as loading control. ( B ) qPCR analysis for indicated back-splice junctions in DHX9-depleted cells (HEK293T). CircBRIP1, known to be regulated by DHX9 ( 29 ), was used as positive control. RPL34 was used to normalize RNA content in parallel RT-PCR reactions omitting RNAse R treatment (mean ± SD, n = 3; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; unpaired t-test). ( C ) Schematic representation of splicing and back-splicing events that generate the two exon 8-containing SMN circRNA, as indicated in the figure. Exons are represented as boxes and introns as lines. In blue and in white are indicated the coding and the non-coding exons, respectively. Shown below is the nucleotide sequence of SMN exon 8 and in red is indicated the cryptic 5′ splice site (c 5′SS) involved in the back-splicing event with the 3′ splice site of exon 6. The sequence of ASO-E8 is highlighted in the rectangle. pA stands for poly A site and 3′dwr stands for 3′ downstream region indicating a cryptic exon of 98 bp involved in circularization and located downstream from exon 8 of SMN . ( D ) Representative images of RT-PCR analysis for the indicated SMN2 circRNAs in ASO treated cells (GM03813). ASO-C is a control morpholino. ( E ) Representative images (left panel) and densitometry (right panel) of western blot for SMN and GEMIN 2 in ASO treated cells (GM03813). Actin was evaluated as loading control (mean ± SD, n = 5; * P ≤ 0.05, ns not significant; unpaired t -test).
Figure Legend Snippet: Interfering with SMN circRNAs biogenesis affects the expression of SMN protein. ( A ) Western blot analysis of DHX9 in DHX9-depleted cells (HEK293T). Actin was evaluated as loading control. ( B ) qPCR analysis for indicated back-splice junctions in DHX9-depleted cells (HEK293T). CircBRIP1, known to be regulated by DHX9 ( 29 ), was used as positive control. RPL34 was used to normalize RNA content in parallel RT-PCR reactions omitting RNAse R treatment (mean ± SD, n = 3; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; unpaired t-test). ( C ) Schematic representation of splicing and back-splicing events that generate the two exon 8-containing SMN circRNA, as indicated in the figure. Exons are represented as boxes and introns as lines. In blue and in white are indicated the coding and the non-coding exons, respectively. Shown below is the nucleotide sequence of SMN exon 8 and in red is indicated the cryptic 5′ splice site (c 5′SS) involved in the back-splicing event with the 3′ splice site of exon 6. The sequence of ASO-E8 is highlighted in the rectangle. pA stands for poly A site and 3′dwr stands for 3′ downstream region indicating a cryptic exon of 98 bp involved in circularization and located downstream from exon 8 of SMN . ( D ) Representative images of RT-PCR analysis for the indicated SMN2 circRNAs in ASO treated cells (GM03813). ASO-C is a control morpholino. ( E ) Representative images (left panel) and densitometry (right panel) of western blot for SMN and GEMIN 2 in ASO treated cells (GM03813). Actin was evaluated as loading control (mean ± SD, n = 5; * P ≤ 0.05, ns not significant; unpaired t -test).

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Positive Control, Reverse Transcription Polymerase Chain Reaction, Sequencing, Allele-specific Oligonucleotide

Sam68 binds Alu -rich introns in SMN and promotes pre-mRNA circularization. ( A ) Graphic representation of the human SMN locus. Exons are indicated with black boxes and introns with lines. Colored arrows represent the identified conserved motifs within Alu elements. Their orientation is indicated by the direction of the arrow. The nucleotide sequences of the three motifs are indicated. ( B ) Predicted binding sites for Sam68, hnRNP F/H and TIA1 were identified (SpliceAid: database of RNA-Splicing Proteins binding). ( C ) Western blot analysis for indicated proteins in Sam68-, hnRNP F/H- and TIA1-depleted cells (HEK293T). Actin was evaluated as loading control. ( D ) qPCR analysis for indicated back-splice junctions in Sam68-, hnRNP F/H- and TIA1-depleted cells (HEK293T). RPL34 was used to normalize for RNA content in parallel RT-PCR reactions omitting RNAse R treatment (mean ± SD, n = 3/5; ∗p ≤ 0.05, ∗∗p ≤ 0.01, *** P ≤ 0.001; unpaired t -test). (E, F) CLIP assay of Sam68 (E, F), hnRNP F/H ( E ) and TIA-1 ( E ) binding to the SMN pre-mRNA. HEK293T cells were UV-crosslinked and immunoprecipitated with control IgGs or antibodies, as indicated. The bar graph shows qPCR signals amplified in CLIP assays expressed as enrichment versus IgG. The value of IgGs was set as 1. The upper panel shows a schematic representation of SMN gene structure and the Alu -overlapping regions amplified by qPCR are indicated with black arrows. The white arrow in panel E indicates the amplified region across exon 2b–intron 2b junction that does not contain Alu s (mean ± SD, n = 6; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; unpaired t -test). ( G ) qPCR analysis for the indicated SMN2 back-splice junctions in cerebellum of adult Sam68 wild type (Sam68 +/+ ) and knockout mice (Sam68 −/− ) harboring the human transgene SMN2++ . Murine L34 (mL34) was used to normalize for RNA content (mean ± SD, n = 3/6; * P ≤ 0.05, ** P ≤ 0.01; unpaired t -test).
Figure Legend Snippet: Sam68 binds Alu -rich introns in SMN and promotes pre-mRNA circularization. ( A ) Graphic representation of the human SMN locus. Exons are indicated with black boxes and introns with lines. Colored arrows represent the identified conserved motifs within Alu elements. Their orientation is indicated by the direction of the arrow. The nucleotide sequences of the three motifs are indicated. ( B ) Predicted binding sites for Sam68, hnRNP F/H and TIA1 were identified (SpliceAid: database of RNA-Splicing Proteins binding). ( C ) Western blot analysis for indicated proteins in Sam68-, hnRNP F/H- and TIA1-depleted cells (HEK293T). Actin was evaluated as loading control. ( D ) qPCR analysis for indicated back-splice junctions in Sam68-, hnRNP F/H- and TIA1-depleted cells (HEK293T). RPL34 was used to normalize for RNA content in parallel RT-PCR reactions omitting RNAse R treatment (mean ± SD, n = 3/5; ∗p ≤ 0.05, ∗∗p ≤ 0.01, *** P ≤ 0.001; unpaired t -test). (E, F) CLIP assay of Sam68 (E, F), hnRNP F/H ( E ) and TIA-1 ( E ) binding to the SMN pre-mRNA. HEK293T cells were UV-crosslinked and immunoprecipitated with control IgGs or antibodies, as indicated. The bar graph shows qPCR signals amplified in CLIP assays expressed as enrichment versus IgG. The value of IgGs was set as 1. The upper panel shows a schematic representation of SMN gene structure and the Alu -overlapping regions amplified by qPCR are indicated with black arrows. The white arrow in panel E indicates the amplified region across exon 2b–intron 2b junction that does not contain Alu s (mean ± SD, n = 6; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; unpaired t -test). ( G ) qPCR analysis for the indicated SMN2 back-splice junctions in cerebellum of adult Sam68 wild type (Sam68 +/+ ) and knockout mice (Sam68 −/− ) harboring the human transgene SMN2++ . Murine L34 (mL34) was used to normalize for RNA content (mean ± SD, n = 3/6; * P ≤ 0.05, ** P ≤ 0.01; unpaired t -test).

Techniques Used: Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Cross-linking Immunoprecipitation, Immunoprecipitation, Amplification, Knock-Out, Mouse Assay

Inverted repeat Alu s negatively impact on SMN transcript processing. ( A ) qPCR analyses of the ratio between distal and proximal pre-mRNA region of human (h SMN2 ) and mouse (m Smn ) SMN gene (mean ± SD, n = 5; *** P ≤ 0.001, unpaired t -test). The analysis was performed in hepatocytes isolated from liver of wild type mice (P1) harboring the human SMN2 transgene. ( B ) Box plots representing the density of the Alu elements in Alu -containing coding (C), non coding (NC) genes and in genes with undetermined function (Und). Boxplots were performed using R (v3.2.3). The red circle highlights the position occupied by SMN genes and other colored circles indicate the position occupied by high- ( MARVELD2 and GOT2 ) and low- Alu ( CTDSP2 and POLI ) genes. ( C ) Bar graphs showing results of qPCR analyses of the ratio between distal and proximal pre-mRNA region of high- and low- Alu genes, as indicated (mean ± SD, n = 3). ( D ) Bar graphs showing results of qPCR analyses of the ratio between distal and proximal pre-mRNA region of SMN and low- Alu genes, as indicated. The analysis was performed by comparing human HEK293T and murine hepatocyte cell lines (mean ± SD, n = 3). ( E ) Western blot analysis of DHX9 in total extracts of DHX9-depleted cells (HEK293T). Actin was evaluated as loading control. ( F ) qPCR analyses of the ratio between distal and proximal pre-mRNA region of human SMN gene in DHX9-depleted cells (HEK293T) (mean ± SD, n = 5; * P ≤ 0.05; unpaired t -test). ( G ) A workflow for detection of BrU-labeled nascent RNAs. HEK293T cells silenced or not for DHX9 were treated with DRB for 6 h to block transcription and BrU-labeled newly synthesized total RNAs were collected 1 h after DRB removal. BrU-labeled nascent RNAs were purified by anti-BrdU antibody and subjected to RT-qPCR analysis. ( H ) qPCR analysis of nascent SMN transcripts labeled with BrU in DHX9-depleted cells (HEK293T). Novel transcripts were immunoprecipitated by using specific anti-BrdU antibody and analyzed with the indicated primers (left side). Cells not treated with BrU were used as control of the immunoprecipitation. The graph represents the ratio between mRNA versus pre-mRNA of specific SMN regions. The value of siCtrl cells was set as reference (mean ± SD, n = 3; ∗p ≤ 0.05, ** P ≤ 0.01; unpaired t-test). ( I ) Representative images (left panel) and densitometry (right panel) of the western blot analyses of DHX9, SMN and GEMIN 2 expression in DHX9-depleted cells (HEK293T). Actin was evaluated as loading control (mean ± SD, n = 3; * P ≤ 0.05, ** P ≤ 0.01, ns not significant; unpaired t -test).
Figure Legend Snippet: Inverted repeat Alu s negatively impact on SMN transcript processing. ( A ) qPCR analyses of the ratio between distal and proximal pre-mRNA region of human (h SMN2 ) and mouse (m Smn ) SMN gene (mean ± SD, n = 5; *** P ≤ 0.001, unpaired t -test). The analysis was performed in hepatocytes isolated from liver of wild type mice (P1) harboring the human SMN2 transgene. ( B ) Box plots representing the density of the Alu elements in Alu -containing coding (C), non coding (NC) genes and in genes with undetermined function (Und). Boxplots were performed using R (v3.2.3). The red circle highlights the position occupied by SMN genes and other colored circles indicate the position occupied by high- ( MARVELD2 and GOT2 ) and low- Alu ( CTDSP2 and POLI ) genes. ( C ) Bar graphs showing results of qPCR analyses of the ratio between distal and proximal pre-mRNA region of high- and low- Alu genes, as indicated (mean ± SD, n = 3). ( D ) Bar graphs showing results of qPCR analyses of the ratio between distal and proximal pre-mRNA region of SMN and low- Alu genes, as indicated. The analysis was performed by comparing human HEK293T and murine hepatocyte cell lines (mean ± SD, n = 3). ( E ) Western blot analysis of DHX9 in total extracts of DHX9-depleted cells (HEK293T). Actin was evaluated as loading control. ( F ) qPCR analyses of the ratio between distal and proximal pre-mRNA region of human SMN gene in DHX9-depleted cells (HEK293T) (mean ± SD, n = 5; * P ≤ 0.05; unpaired t -test). ( G ) A workflow for detection of BrU-labeled nascent RNAs. HEK293T cells silenced or not for DHX9 were treated with DRB for 6 h to block transcription and BrU-labeled newly synthesized total RNAs were collected 1 h after DRB removal. BrU-labeled nascent RNAs were purified by anti-BrdU antibody and subjected to RT-qPCR analysis. ( H ) qPCR analysis of nascent SMN transcripts labeled with BrU in DHX9-depleted cells (HEK293T). Novel transcripts were immunoprecipitated by using specific anti-BrdU antibody and analyzed with the indicated primers (left side). Cells not treated with BrU were used as control of the immunoprecipitation. The graph represents the ratio between mRNA versus pre-mRNA of specific SMN regions. The value of siCtrl cells was set as reference (mean ± SD, n = 3; ∗p ≤ 0.05, ** P ≤ 0.01; unpaired t-test). ( I ) Representative images (left panel) and densitometry (right panel) of the western blot analyses of DHX9, SMN and GEMIN 2 expression in DHX9-depleted cells (HEK293T). Actin was evaluated as loading control (mean ± SD, n = 3; * P ≤ 0.05, ** P ≤ 0.01, ns not significant; unpaired t -test).

Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Mouse Assay, Western Blot, Labeling, Blocking Assay, Synthesized, Purification, Quantitative RT-PCR, Immunoprecipitation, Expressing

2) Product Images from "SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy"

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201502059

Skeletal muscle atrophy is ameliorated in the SMAΔ7/ Sam68 −/− mice. (A) Hematoxylin and eosin staining of triceps muscle sections from 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Bar, 100 µm. (B) Frequency distribution of muscle fiber areas (at least 2,000 for each genotype) of the experimental animals described in A. The colored lines and dotted line (non-SMA mice) represent the median value for each genotype: non-SMA mice ( n = 3) median size = 531 µm 2 and fiber size (mean ± SEM) = 568.76 ± 5.8 µm 2 ; non-SMA/ Sam68 −/− mice ( n = 3) median size = 557 µm 2 and fiber size (mean ± SEM) = 586.60 ± 5.7 µm 2 ; SMAΔ7/ Sam68 +/+ median size = 327 µm 2 and fiber size (mean ± SEM) = 342.32 ± 3.1 µm 2 ; and SMAΔ7/ Sam68 −/− median size = 486 µm 2 and fiber size (mean ± SEM) = 503.59 ± 4.6 µm 2 . Statistical analysis of the median values among groups was performed by one-way ANOVA test followed by Dunn’s multiple comparison posttest. (C) qPCR analysis of the expression of atrophy-related genes in quadriceps and lower leg muscles of mice described in B. Values (mean ± SD; n = 4) were normalized with Actin mRNA. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest. *, P
Figure Legend Snippet: Skeletal muscle atrophy is ameliorated in the SMAΔ7/ Sam68 −/− mice. (A) Hematoxylin and eosin staining of triceps muscle sections from 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Bar, 100 µm. (B) Frequency distribution of muscle fiber areas (at least 2,000 for each genotype) of the experimental animals described in A. The colored lines and dotted line (non-SMA mice) represent the median value for each genotype: non-SMA mice ( n = 3) median size = 531 µm 2 and fiber size (mean ± SEM) = 568.76 ± 5.8 µm 2 ; non-SMA/ Sam68 −/− mice ( n = 3) median size = 557 µm 2 and fiber size (mean ± SEM) = 586.60 ± 5.7 µm 2 ; SMAΔ7/ Sam68 +/+ median size = 327 µm 2 and fiber size (mean ± SEM) = 342.32 ± 3.1 µm 2 ; and SMAΔ7/ Sam68 −/− median size = 486 µm 2 and fiber size (mean ± SEM) = 503.59 ± 4.6 µm 2 . Statistical analysis of the median values among groups was performed by one-way ANOVA test followed by Dunn’s multiple comparison posttest. (C) qPCR analysis of the expression of atrophy-related genes in quadriceps and lower leg muscles of mice described in B. Values (mean ± SD; n = 4) were normalized with Actin mRNA. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest. *, P

Techniques Used: Mouse Assay, Staining, Real-time Polymerase Chain Reaction, Expressing

Sam68 deletion rescues SMN2 splicing and expression in SMAΔ7 tissues. (A) qPCR analysis of exon 7–containing SMN2 transgene transcripts normalized to constant exon 6 in tissues of 10-dpp SMAΔ7/ Sam68 +/+ and SMAΔ7/ Sam68 −/− mice. Bar graph represents mean ± SD. n = 3. The p-value was determined by two-tailed t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Western blot analysis of SMN protein expression in the indicated tissues of 10-dpp SMAΔ7/ Sam68 +/+ (wt) or SMAΔ7/ Sam68 −/− (ko) mice. Actin was used as a loading control. Quantification of the SMN band is shown at the bottom. Each point value represents the mean ± SD. n = 2. Cereb, cerebellum; ko, knockout; S.cord, spinal cord; wt, wild type.
Figure Legend Snippet: Sam68 deletion rescues SMN2 splicing and expression in SMAΔ7 tissues. (A) qPCR analysis of exon 7–containing SMN2 transgene transcripts normalized to constant exon 6 in tissues of 10-dpp SMAΔ7/ Sam68 +/+ and SMAΔ7/ Sam68 −/− mice. Bar graph represents mean ± SD. n = 3. The p-value was determined by two-tailed t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Western blot analysis of SMN protein expression in the indicated tissues of 10-dpp SMAΔ7/ Sam68 +/+ (wt) or SMAΔ7/ Sam68 −/− (ko) mice. Actin was used as a loading control. Quantification of the SMN band is shown at the bottom. Each point value represents the mean ± SD. n = 2. Cereb, cerebellum; ko, knockout; S.cord, spinal cord; wt, wild type.

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

3) Product Images from "PRDM14 is expressed in germ cell tumors with constitutive overexpression altering human germline differentiation and proliferation"

Article Title: PRDM14 is expressed in germ cell tumors with constitutive overexpression altering human germline differentiation and proliferation

Journal: Stem cell research

doi: 10.1016/j.scr.2017.12.016

PRDM14 expression in testicular and intracranial germ cell tumors A. Western blot of nuclear (5 μg) and cytoplasmic (5 μg) extracts from embryonal carcinoma cell (ECC) lines, Ntera2 (NT) and GCT27. Blots probed for PRDM14 (64 kD), OCT4 (45 kD), and beta-actin (42 kD). B. Immunofluorescence of ECC line, GCT27, showing staining for PRDM14/OCT4, top panel. Immunofluorescence of paraffin section of a seminoma tumor sample, showing staining for PRDM14/OCT4, bottom panel. Scale bars, 15 μm. C. Immunohistochemistry of paraffin section of tumor sample containing corresponding GCNIS portion of the tumor, staining for PRDM14, and GCNIS markers OCT4 and PLAP. Scale bars, 15 μm. D. Immunohistochemistry of paraffin section of mixed germ cell tumor, corresponding to embryonal carcinoma, staining for PRDM14, SOX2 and SOX17, top panel. Magnified images of cells demarked in yellow box in top panel. Scale bars, 15 μm (top panel). 5 μm (bottom panel). E. Immunohistochemistry staining of intracranial germinoma, yolk sac and teratoma, for PRDM14. Scale bars, 20 μm.
Figure Legend Snippet: PRDM14 expression in testicular and intracranial germ cell tumors A. Western blot of nuclear (5 μg) and cytoplasmic (5 μg) extracts from embryonal carcinoma cell (ECC) lines, Ntera2 (NT) and GCT27. Blots probed for PRDM14 (64 kD), OCT4 (45 kD), and beta-actin (42 kD). B. Immunofluorescence of ECC line, GCT27, showing staining for PRDM14/OCT4, top panel. Immunofluorescence of paraffin section of a seminoma tumor sample, showing staining for PRDM14/OCT4, bottom panel. Scale bars, 15 μm. C. Immunohistochemistry of paraffin section of tumor sample containing corresponding GCNIS portion of the tumor, staining for PRDM14, and GCNIS markers OCT4 and PLAP. Scale bars, 15 μm. D. Immunohistochemistry of paraffin section of mixed germ cell tumor, corresponding to embryonal carcinoma, staining for PRDM14, SOX2 and SOX17, top panel. Magnified images of cells demarked in yellow box in top panel. Scale bars, 15 μm (top panel). 5 μm (bottom panel). E. Immunohistochemistry staining of intracranial germinoma, yolk sac and teratoma, for PRDM14. Scale bars, 20 μm.

Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining, Paraffin Section, Immunohistochemistry

4) Product Images from "The Multi-Copy Mouse Gene Sycp3-Like Y-Linked (Sly) Encodes an Abundant Spermatid Protein That Interacts with a Histone Acetyltransferase and an Acrosomal Protein 1"

Article Title: The Multi-Copy Mouse Gene Sycp3-Like Y-Linked (Sly) Encodes an Abundant Spermatid Protein That Interacts with a Histone Acetyltransferase and an Acrosomal Protein 1

Journal:

doi: 10.1095/biolreprod.108.075382

Western blot analysis of SLY1. A ) Western blot analysis of multiple tissues from an adult mouse. The anti-SLY1 antibody detects a testis-specific protein of approximately 40 kDa (arrow) that is presumed to be SLY1. An antibody against ACTIN was
Figure Legend Snippet: Western blot analysis of SLY1. A ) Western blot analysis of multiple tissues from an adult mouse. The anti-SLY1 antibody detects a testis-specific protein of approximately 40 kDa (arrow) that is presumed to be SLY1. An antibody against ACTIN was

Techniques Used: Western Blot

Related Articles

Western Blot:

Article Title: Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation
Article Snippet: .. Antibodies The following antibodies were used for western blotting (WB) and immunofluorescence (IF): mouse anti-beta-actin (Santa Cruz, catalogue number sc-47778; WB 1:2000), mouse anti-HA (ENZO, catalogue number ENZ-ABS120-0200; WB 1:1000), mouse anti-transferrin receptor (Invitrogen, catalogue number 13–6800; WB 1:1000), rabbit anti-Stim1 (Cell Signalling Technology, catalogue number 5668S (D88E10); WB 1:2000), rabbit anti-Orai1 (Sigma Aldrich, catalogue number O8264; WB 1:2500), goat anti-myc tag (Abcam, catalogue number ab9132; IF 1:2000), rabbit anti-RHBDL2 (Proteintech, catalogue number 12467-1-AP; WB 1:250 – only detected RHBDL2 in HaCaT lysates), rabbit anti-V5 tag (Cell Signalling Technology, catalogue number 13202S; WB and IF 1:2000). .. Corresponding species-specific HRP or fluorescently coupled secondary antibodies were used from Santa Cruz and Cell Signaling (WB) or Invitrogen (IF).

Article Title: Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling
Article Snippet: .. Antibodies The following antibodies were used: mouse anti-beta-actin (Santa Cruz, catalogue number sc-47778; WB 1:2000), rabbit anti-TACE/ADAM17 (Abcam, catalogue number ab39161; WB 1:2000), rabbit anti-HA (Santa-Cruz, catalogue number sc-805; IF 1:1000), mouse anti-HA (ENZO, catalogue number ENZ-ABS120-0200; PM IP 1:1000), mouse anti-transferrin receptor (Invitrogen, catalogue number 13–6800; 1:1000), rabbit anti-phosphoserine (Invitrogen, catalogue number 618100; WB 1:1000), rat HA-HRP, clone 3F10 (Roche, catalogue number 12013819001; WB 1:2000), mouse anti-p97 (Pierce/Thermo, catalogue number MA1-21412; WB 1:1000), rabbit anti-pan14-3-3 (Cell Signalling, catalogue number 8312; WB 1:1000), mouse anti-GM130 (BD Transduction labs, catalogue number 610823; IF 1:1000), rabbit anti-pan-cadherin (Cell Signalling, catalogue number 4068S; WB 1:1000), rabbit anti-ADAM10 (Abcam, catalogue number ab1997; WB 1:1000), rabbit anti-ERK1/2 (Cell Signalling, catalogue number 9102, WB 1:1000), rabbit anti-pERK1/2 (Cell Signalling, catalogue number 4377, WB 1:1000). .. Corresponding species-specific HRP or fluorescently coupled secondary antibodies were used from Santa Cruz and Cell Signaling (WB) or Invitrogen (IF).

Article Title: Zika virus infection induces MiR34c expression in glioblastoma stem cells: new perspectives for brain tumor treatments
Article Snippet: .. The antibodies for WB were used at the condition suggested by the suppliers: rabbit anti-NOTCH-1 (Ab27526, 1/500, Santa Cruz Biotechnology, Texas, USA), rabbit anti-p73 (Ab189896, 1/1000, Abcam), rabbit anti-Bcl2 (Ab185002, 1/500, Abcam), rabbit anti-human NUMB (ab-14140, 1/1000, Abcam), goat anti-AKT (C-20) (1/500, sc-1618, Santa Cruz), rabbit anti-Phospho-Akt-Ser-473 (#9271 1/500, Cell Signaling), and mouse anti-beta-actin (sc-81178, 1/1000, Santa Cruz Biotechnology). .. Statistical analysis Expression values of numb mRNA in normal tissues and in glioma samples, distributed by histology classification, with a cutoff equal to the median (p -value with Bonferroni) were downloaded from GlioVis Data Portal .

other:

Article Title: Genetic interaction implicates iRhom2 in the regulation of EGF receptor signalling in mice
Article Snippet: Antibodies The following antibodies were used: Goat anti-mouse amphiregulin (R & D Systems, catalogue number AF989; 1:1000), Mouse anti-nonphospho-Tyr1173-EGFR (Millipore, catalogue number 05-484; 1:1000), mouse anti-beta-actin (Santa Cruz, catalogue number sc-47778; 1:2000), rabbit anti-TACE/ADAM17 (Abcam, catalogue number ab39161; 1:2000), rabbit anti-HA (Santa-Cruz, catalogue number sc-805; 1:2000) and mouse anti-transferrin receptor (Invitrogen, catalogue number 13-6800; 1:1000).

Immunofluorescence:

Article Title: Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation
Article Snippet: .. Antibodies The following antibodies were used for western blotting (WB) and immunofluorescence (IF): mouse anti-beta-actin (Santa Cruz, catalogue number sc-47778; WB 1:2000), mouse anti-HA (ENZO, catalogue number ENZ-ABS120-0200; WB 1:1000), mouse anti-transferrin receptor (Invitrogen, catalogue number 13–6800; WB 1:1000), rabbit anti-Stim1 (Cell Signalling Technology, catalogue number 5668S (D88E10); WB 1:2000), rabbit anti-Orai1 (Sigma Aldrich, catalogue number O8264; WB 1:2500), goat anti-myc tag (Abcam, catalogue number ab9132; IF 1:2000), rabbit anti-RHBDL2 (Proteintech, catalogue number 12467-1-AP; WB 1:250 – only detected RHBDL2 in HaCaT lysates), rabbit anti-V5 tag (Cell Signalling Technology, catalogue number 13202S; WB and IF 1:2000). .. Corresponding species-specific HRP or fluorescently coupled secondary antibodies were used from Santa Cruz and Cell Signaling (WB) or Invitrogen (IF).

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    Santa Cruz Biotechnology mouse anti β actin mab
    ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 <t>β−Actin</t> copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value
    Mouse Anti β Actin Mab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology mouse anti human β actin
    mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. <t>β-actin</t> was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P
    Mouse Anti Human β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology polyclonal actin antibody
    Ferulic acid (FA) inhibits amyloidogenic amyloid precursor protein (APP) metabolism in SweAPP N2a cells by modulating β-site APP cleaving enzyme 1 (BACE1) expression and activity. (A) Amyloid-β (Aβ) 1–40 and Aβ 1–42 species were separately measured in cell supernatants from SweAPP N2a cells by sandwich ELISAs. (B) Inhibition of amyloidogenic APP processing in SweAPP N2a cells treated with various doses of FA. Western blots using an amino-terminal Aβ 1–17 monoclonal antibody (mAb 6E10), a carboxyl-terminal APP <t>polyclonal</t> antibody (pAb C-APP), or a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1) show holo-APP, carboxyl-terminal fragment generated by amyloidogenic APP cleavage (C99, β-CTF), and BACE1, respectively. Actin is included as an internal reference control, and ratiometric densitometry data are shown below each lane. (C) Densitometry results are shown as ratio of C99 to actin at various FA treatment doses. (D) Densitometry results are shown as ratio of BACE1 to actin at various FA treatment doses. (E) Quantitative real-time PCR results for BACE1 mRNA levels at various FA treatment doses are shown in arbitrary units, and BACE1 relative abundance is depicted on the y-axis. (F) Cell-free BACE1 activity assay results are displayed, and % of BACE1 activity is shown on the y-axis. (G) Lactate dehydrogenase (LDH) release assay results are shown for SweAPP N2a cells treated with 0 to 12.5 µM of FA. All statistical comparisons are vs. 0 µM of FA, and similar results were observed in 3–4 independent experiments.
    Polyclonal Actin Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value

    Journal: Scientific Reports

    Article Title: An allelic variant in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression of the two genes

    doi: 10.1038/s41598-018-28799-8

    Figure Lengend Snippet: ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value

    Article Snippet: The membranes were incubated ON with mouse anti-ERAP1 mAb antibody (clone B-10, sc-271823 SantaCruz), mouse anti-ERAP2 mAb (clone 3F5, MAB 3830 R & D Systems) and mouse anti-β-Actin mAb (clone C4, sc-477778 SantaCruz).

    Techniques: Inhibition

    Polyubiquitinated p53 species detected in U2OS cells titrated with LMP1 . Western blot detection of transfected LMP1, endogenous p53 protein levels and detection of ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP1 (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increased polyubiquitinated p53 protein was seen as high molecular weight aggregates in LMP1 expressing cells.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: Polyubiquitinated p53 species detected in U2OS cells titrated with LMP1 . Western blot detection of transfected LMP1, endogenous p53 protein levels and detection of ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP1 (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increased polyubiquitinated p53 protein was seen as high molecular weight aggregates in LMP1 expressing cells.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Transfection, Immunoprecipitation, Molecular Weight, Expressing

    Dominant-negative Akt (DN-Akt) does not restore p53 protein levels . Western blot detection of LMP1, Dominant-negative Akt (DN-Akt) and p53 protein levels in U2OS cells transfected with fixed amount of LMP1 (0.5 μg) and titrated with DN-Akt (0.05 - 4.0 μg). β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increasing amounts of DN-Akt (detected by anti-Akt antibodies) did not rescue reduction of p53 protein levels by LMP1.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: Dominant-negative Akt (DN-Akt) does not restore p53 protein levels . Western blot detection of LMP1, Dominant-negative Akt (DN-Akt) and p53 protein levels in U2OS cells transfected with fixed amount of LMP1 (0.5 μg) and titrated with DN-Akt (0.05 - 4.0 μg). β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. Increasing amounts of DN-Akt (detected by anti-Akt antibodies) did not rescue reduction of p53 protein levels by LMP1.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Dominant Negative Mutation, Western Blot, Transfection

    LMP1, but not LMP2A or EBNA1, reduces p53 protein levels . (A) Western blot analysis of the effects of LMP1, LMP2A and EBNA1 on the expression levels of endogenous p53 protein in U2OS cells. Transfection of LMP1 reduced the level of p53 protein. (B) Co-transfection of LMP1 abolished p53 protein level in HONE1 NPC cells transiently transfected with p53. A slight reduction in the exogenous p53 protein level in LMP2A co-transfected in HONE1 NPC cells setting but not in U2OS cells in (A). ( C ) EBV-negative HONE1 and EBV-positive HONE Akata (HA) cells were transfected with p53 construct. p53 protein levels were determined in the Actinomycin-D induced and uninduced state. EBV-positive HA NPC cells have lower levels of p53 protein in comparison with EBV-negative HONE NPC cells. β-actin was served as a loading control in all the three experiments. Representative blots were quantitated using Image J.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: LMP1, but not LMP2A or EBNA1, reduces p53 protein levels . (A) Western blot analysis of the effects of LMP1, LMP2A and EBNA1 on the expression levels of endogenous p53 protein in U2OS cells. Transfection of LMP1 reduced the level of p53 protein. (B) Co-transfection of LMP1 abolished p53 protein level in HONE1 NPC cells transiently transfected with p53. A slight reduction in the exogenous p53 protein level in LMP2A co-transfected in HONE1 NPC cells setting but not in U2OS cells in (A). ( C ) EBV-negative HONE1 and EBV-positive HONE Akata (HA) cells were transfected with p53 construct. p53 protein levels were determined in the Actinomycin-D induced and uninduced state. EBV-positive HA NPC cells have lower levels of p53 protein in comparison with EBV-negative HONE NPC cells. β-actin was served as a loading control in all the three experiments. Representative blots were quantitated using Image J.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Expressing, Transfection, Cotransfection, Construct

    Only monoubiquitinated p53 species detected in U2OS titrated with LMP2A . Western blot detection of transfected LMP2A-HA, endogenous p53 protein levels and ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP2A-HA (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. No polyubiquitinated p53 protein was seen in LMP2A expressing cells.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: Only monoubiquitinated p53 species detected in U2OS titrated with LMP2A . Western blot detection of transfected LMP2A-HA, endogenous p53 protein levels and ubiquitinated p53 species in U2OS cells transiently transfected with titrated LMP2A-HA (0.05 - 4.0 μg). For the ubiquitination experiment, p53 protein was immunoprecipitated from the cell lysate with anti-p53 mouse monoclonal antibody (DO-1) and then immunoblotted with anti-ubiquitin rabbit polyclonal antibody. β-actin was served as a loading control in the experiment. Representative blots were quantitated using Image J. No polyubiquitinated p53 protein was seen in LMP2A expressing cells.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Transfection, Immunoprecipitation, Expressing

    PI3K-Akt inhibitor LY294002 does not restore p53 protein levels . (A) Western blot analysis of the effects of PI3K inhibitor LY294002 on the endogenous p53 protein levels in U2OS cells. The cells were treated overnight, with 25 μM LY294002 or vehicle alone (DMSO). Treatment by LY294002 did not rescue the reduction of p53 protein levels by LMP1. (B) Western blot analysis of the effects of PI3K inhibitor LY294002 on the transfected p53 protein levels in CNE1-LMP1 stable cell line. The cells were treated overnight with 25 μM LY294002 or vehicle alone (DMSO). β-actin was served as a loading control in both the experiments. Representative blots were quantitated using Image J. Treatment with LY294002 did not rescue the reduction of p53 protein levels by LMP1.

    Journal: BMC Research Notes

    Article Title: Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

    doi: 10.1186/1756-0500-4-551

    Figure Lengend Snippet: PI3K-Akt inhibitor LY294002 does not restore p53 protein levels . (A) Western blot analysis of the effects of PI3K inhibitor LY294002 on the endogenous p53 protein levels in U2OS cells. The cells were treated overnight, with 25 μM LY294002 or vehicle alone (DMSO). Treatment by LY294002 did not rescue the reduction of p53 protein levels by LMP1. (B) Western blot analysis of the effects of PI3K inhibitor LY294002 on the transfected p53 protein levels in CNE1-LMP1 stable cell line. The cells were treated overnight with 25 μM LY294002 or vehicle alone (DMSO). β-actin was served as a loading control in both the experiments. Representative blots were quantitated using Image J. Treatment with LY294002 did not rescue the reduction of p53 protein levels by LMP1.

    Article Snippet: Antibodies Primary antibodies used in this work were anti-p53 mouse monoclonal antibody (DO-1) (Santa Cruz, California, USA) at 1:1000 dilution, anti-LMP1 mouse monoclonal antibody (DAKO, Denmark) at 1:500 dilution, rat monoclonal EBV LMP2A, Clone 14B7 (E. Kremmer, Institute for Molecular Immunology, Munich) at 1:50 dilution, anti-Akt rabbit polyclonal antibody (Cell Signalling Technology, Danvers, USA) at 1:1000 dilution, anti-ubiquitin rabbit polyclonal antibody (Sigma, Saint Louis, USA) at 1:100 dilution, and anti-β-actin mouse monoclonal antibody (Santa Cruz, California, USA) at 1:1000 which served as a loading control protein.

    Techniques: Western Blot, Transfection, Stable Transfection

    mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. β-actin was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P

    Journal: International Journal of Molecular Medicine

    Article Title: All-trans retinoic acid alters the expression of the tight junction proteins Claudin-1 and -4 and epidermal barrier function-associated genes in the epidermis

    doi: 10.3892/ijmm.2019.4098

    Figure Lengend Snippet: mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. β-actin was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P

    Article Snippet: TBS buffer containing Tween-20 (TBST) and 5% non-fat milk was used to block non-specific binding at room temperature for 2 h. Following blocking, primary antibodies were incubated overnight at 4°C: Rabbit anti-human Claudin-1 (cat. no. 13050-1-AP; ProteinTech Group, Inc., Chicago, IL, USA; 1:200), goat anti-human Claudin-4 (cat. no. 17664; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200) and mouse anti-human β-actin (cat. no. 47778; Santa Cruz Biotechnology, Inc.; 1:1,000).

    Techniques: Expressing, Incubation, Mouse Assay, Western Blot, Standard Deviation

    Ferulic acid (FA) inhibits amyloidogenic amyloid precursor protein (APP) metabolism in SweAPP N2a cells by modulating β-site APP cleaving enzyme 1 (BACE1) expression and activity. (A) Amyloid-β (Aβ) 1–40 and Aβ 1–42 species were separately measured in cell supernatants from SweAPP N2a cells by sandwich ELISAs. (B) Inhibition of amyloidogenic APP processing in SweAPP N2a cells treated with various doses of FA. Western blots using an amino-terminal Aβ 1–17 monoclonal antibody (mAb 6E10), a carboxyl-terminal APP polyclonal antibody (pAb C-APP), or a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1) show holo-APP, carboxyl-terminal fragment generated by amyloidogenic APP cleavage (C99, β-CTF), and BACE1, respectively. Actin is included as an internal reference control, and ratiometric densitometry data are shown below each lane. (C) Densitometry results are shown as ratio of C99 to actin at various FA treatment doses. (D) Densitometry results are shown as ratio of BACE1 to actin at various FA treatment doses. (E) Quantitative real-time PCR results for BACE1 mRNA levels at various FA treatment doses are shown in arbitrary units, and BACE1 relative abundance is depicted on the y-axis. (F) Cell-free BACE1 activity assay results are displayed, and % of BACE1 activity is shown on the y-axis. (G) Lactate dehydrogenase (LDH) release assay results are shown for SweAPP N2a cells treated with 0 to 12.5 µM of FA. All statistical comparisons are vs. 0 µM of FA, and similar results were observed in 3–4 independent experiments.

    Journal: PLoS ONE

    Article Title: Ferulic Acid Is a Nutraceutical ?-Secretase Modulator That Improves Behavioral Impairment and Alzheimer-like Pathology in Transgenic Mice

    doi: 10.1371/journal.pone.0055774

    Figure Lengend Snippet: Ferulic acid (FA) inhibits amyloidogenic amyloid precursor protein (APP) metabolism in SweAPP N2a cells by modulating β-site APP cleaving enzyme 1 (BACE1) expression and activity. (A) Amyloid-β (Aβ) 1–40 and Aβ 1–42 species were separately measured in cell supernatants from SweAPP N2a cells by sandwich ELISAs. (B) Inhibition of amyloidogenic APP processing in SweAPP N2a cells treated with various doses of FA. Western blots using an amino-terminal Aβ 1–17 monoclonal antibody (mAb 6E10), a carboxyl-terminal APP polyclonal antibody (pAb C-APP), or a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1) show holo-APP, carboxyl-terminal fragment generated by amyloidogenic APP cleavage (C99, β-CTF), and BACE1, respectively. Actin is included as an internal reference control, and ratiometric densitometry data are shown below each lane. (C) Densitometry results are shown as ratio of C99 to actin at various FA treatment doses. (D) Densitometry results are shown as ratio of BACE1 to actin at various FA treatment doses. (E) Quantitative real-time PCR results for BACE1 mRNA levels at various FA treatment doses are shown in arbitrary units, and BACE1 relative abundance is depicted on the y-axis. (F) Cell-free BACE1 activity assay results are displayed, and % of BACE1 activity is shown on the y-axis. (G) Lactate dehydrogenase (LDH) release assay results are shown for SweAPP N2a cells treated with 0 to 12.5 µM of FA. All statistical comparisons are vs. 0 µM of FA, and similar results were observed in 3–4 independent experiments.

    Article Snippet: Membranes were then hybridized for 1 hour at ambient temperature with primary antibodies: an amino-terminal APP polyclonal antibody (1∶400, IBL), a carboxyl-terminal APP polyclonal antibody (1∶500, Merck Millipore), a carboxyl-terminal PS1 monoclonal antibody (PS1-loop; 1∶500, Merck Millipore), a carboxyl-terminal BACE1 polyclonal antibody (1∶400, IBL), an amino-terminal Aβ1–16 monoclonal antibody (82E1; 1∶150, IBL), an amino-terminal Aβ1–17 monoclonal antibody (6E10; 1∶1,000, Merck Millipore), or a polyclonal actin antibody as a loading control (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Activity Assay, Inhibition, Western Blot, Generated, Real-time Polymerase Chain Reaction, Lactate Dehydrogenase Assay

    β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) modulation and inhibition of amyloidogenic APP processing in PSAPP mice treated with ferulic acid (FA). (A) Western blots are shown using an amino (N)-terminal APP polyclonal antibody (pAb N-APP; holo-APP is shown) and a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1). Western blots are also shown using an amino-terminal amyloid-β (Aβ) monoclonal antibody (mAb 82E1), which detects various amyloidogenic APP cleavage fragments including: Aβ monomer and oligomers as well as phospho-C99 (P-C99) and non-phospho-C99 (C99). Actin is shown as a loading control for each blot, and ratiometric densitometry data are shown below each lane. (B) Densitometry analyses are shown for ratios of C-99 or P-C99 to actin. (C) Aβ oligomers in the 2% SDS-soluble brain homogenate fraction were measured by sandwich ELISA. (D) Densitometry analysis is shown for ratio of BACE1 to actin. (E) α- and β-secretase activity assays are shown. Relative fluorescence units are depicted on the y-axis, and reaction time is represented on the x-axis. Representative Western blots for A were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 3) or with FA (PSAPP-FA, n = 3). Data for B-E were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 12) or with FA (PSAPP-FA, n = 12) for 6 months beginning at 6 months of age. All statistical comparisons are between PSAPP-V and PSAPP-FA mice.

    Journal: PLoS ONE

    Article Title: Ferulic Acid Is a Nutraceutical ?-Secretase Modulator That Improves Behavioral Impairment and Alzheimer-like Pathology in Transgenic Mice

    doi: 10.1371/journal.pone.0055774

    Figure Lengend Snippet: β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) modulation and inhibition of amyloidogenic APP processing in PSAPP mice treated with ferulic acid (FA). (A) Western blots are shown using an amino (N)-terminal APP polyclonal antibody (pAb N-APP; holo-APP is shown) and a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1). Western blots are also shown using an amino-terminal amyloid-β (Aβ) monoclonal antibody (mAb 82E1), which detects various amyloidogenic APP cleavage fragments including: Aβ monomer and oligomers as well as phospho-C99 (P-C99) and non-phospho-C99 (C99). Actin is shown as a loading control for each blot, and ratiometric densitometry data are shown below each lane. (B) Densitometry analyses are shown for ratios of C-99 or P-C99 to actin. (C) Aβ oligomers in the 2% SDS-soluble brain homogenate fraction were measured by sandwich ELISA. (D) Densitometry analysis is shown for ratio of BACE1 to actin. (E) α- and β-secretase activity assays are shown. Relative fluorescence units are depicted on the y-axis, and reaction time is represented on the x-axis. Representative Western blots for A were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 3) or with FA (PSAPP-FA, n = 3). Data for B-E were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 12) or with FA (PSAPP-FA, n = 12) for 6 months beginning at 6 months of age. All statistical comparisons are between PSAPP-V and PSAPP-FA mice.

    Article Snippet: Membranes were then hybridized for 1 hour at ambient temperature with primary antibodies: an amino-terminal APP polyclonal antibody (1∶400, IBL), a carboxyl-terminal APP polyclonal antibody (1∶500, Merck Millipore), a carboxyl-terminal PS1 monoclonal antibody (PS1-loop; 1∶500, Merck Millipore), a carboxyl-terminal BACE1 polyclonal antibody (1∶400, IBL), an amino-terminal Aβ1–16 monoclonal antibody (82E1; 1∶150, IBL), an amino-terminal Aβ1–17 monoclonal antibody (6E10; 1∶1,000, Merck Millipore), or a polyclonal actin antibody as a loading control (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Inhibition, Mouse Assay, Western Blot, Sandwich ELISA, Activity Assay, Fluorescence