Structured Review

Santa Cruz Biotechnology anti beta actin
HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules. ( A ) Immunofluorescence of paxillin ( green ), and alpha-actinin ( red ) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI ( blue ) was included to stain the nuclei. Magnification bar: 30 µ. ( B ) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. ( C ) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). ( D ) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. ( E ) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against <t>beta-actin</t> (as loading control). *P
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Images

1) Product Images from "Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells"

Article Title: Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044147

HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules. ( A ) Immunofluorescence of paxillin ( green ), and alpha-actinin ( red ) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI ( blue ) was included to stain the nuclei. Magnification bar: 30 µ. ( B ) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. ( C ) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). ( D ) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. ( E ) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against beta-actin (as loading control). *P
Figure Legend Snippet: HCV infection affects intracellular localization, expression levels, and activities of focal adhesion molecules. ( A ) Immunofluorescence of paxillin ( green ), and alpha-actinin ( red ) by confocal microscopy in Ctrl and HCV Huh7.5.1 cells after 24 hrs from plating. DAPI ( blue ) was included to stain the nuclei. Magnification bar: 30 µ. ( B ) Paxillin and alpha-actinin protein expression levels observed 24 hrs after plating. ( C ) Immunohistochemical analysis of paxillin expression in HCCs and control livers (×200 magnification). ( D ) Total FAK and tyrosine 397 phosphorylated FAK were observed 24 hrs after plating. Immunoblots are representative of at least four independent experiments. ( E ) Immunohistochemical analysis of tyrosine 397 phosphorylated FAK expression in HCCs and control livers (×200 magnification). In left histograms densitometric analysis is reported as fold changes in protein levels respect to the control cells considered as 1 after normalization against beta-actin (as loading control). *P

Techniques Used: Infection, Expressing, Immunofluorescence, Confocal Microscopy, Staining, Immunohistochemistry, Western Blot

HCV affects proliferation, anchorage-independent growth, adhesion and migration of Huh7.5.1 cells. ( A ) RT-PCR for the 5′UTR in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). ( B ) Protein expression levels of HCV core ( upper panel ), HCV NS3 ( middle panel ) and beta-actin (loading control, lower panel ) in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). Immunoblots are representative of at least five independent experiments. ( C ) Proliferation rate was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD ( bars ) of five independent experiments. *P
Figure Legend Snippet: HCV affects proliferation, anchorage-independent growth, adhesion and migration of Huh7.5.1 cells. ( A ) RT-PCR for the 5′UTR in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). ( B ) Protein expression levels of HCV core ( upper panel ), HCV NS3 ( middle panel ) and beta-actin (loading control, lower panel ) in control (Ctrl) and HCV-infected Huh7.5.1 cells (HCV). Immunoblots are representative of at least five independent experiments. ( C ) Proliferation rate was evaluated as incorporation of BrdU performed at three different time points: 0, 6 and 24 hrs. Quantitative data of the analysis of BrdU incorporation was converted in unit of induction with respect to the Ctrl considered as 1. Histograms are the mean value ±SD ( bars ) of five independent experiments. *P

Techniques Used: Migration, Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, Western Blot, BrdU Incorporation Assay

2) Product Images from "Quercetin-induced apoptosis prevents EBV infection"

Article Title: Quercetin-induced apoptosis prevents EBV infection

Journal: Oncotarget

doi:

Effects of quercetin or isoliquiritigenin on signal transduction Cignal finder reporter assay was conducted to determine what signaling pathways inSNU719 cells are affected by treatments of quercetin or isoliquiritigenin. SNU719 cells were treated 62 μM quercetin or 45 μM isoliquiritigenin for 48 h and subjected to Cignal finder reporter assay. Each reporter is a luciferase construct in which a transcriptional factor specific to a signaling pathway was cloned. Signaling pathways affected by either quercetin or isoliquiritigenin were chosen when their fold changes were immensely higher or lower than negative and positive reporter signals (shaded by red box). Reporters induced by either quercetin or isoliquiritigenin were in blue box and reporters suppressed by either quercetin or isoliquiritigenin were in green box. ERE, HSR, SRE, AP1 reporters were upregulated by quercetin, and these reporters are specific to estrogen pathway, heat shock response pathway, MAPK/ERK pathway and MARK/JNK pathway, respectively. GRE, MTF1, GAS, LXR, SMAD reporters were downregulated by quercetin, and these reporters are specific to glucocorticoid pathway, heavy metal stress pathway, interferon gamma pathway, liver X receptor pathway and TGF-beta pathway, respectively. AARE, SRE, AP1, PPAR, RXR reporters were upregulated by isoliquiritigenin, and these reporters are specific to amino acid deprivation response pathway, MAPK/ERK pathway, MAPK/JNK pathway, PPAR pathway and retinoid X receptor pathway, respectively. IFR1 reporter was downregulated by isoliquiritigenin and this reporter is specific to interferon regulatory factor 1 pathway.
Figure Legend Snippet: Effects of quercetin or isoliquiritigenin on signal transduction Cignal finder reporter assay was conducted to determine what signaling pathways inSNU719 cells are affected by treatments of quercetin or isoliquiritigenin. SNU719 cells were treated 62 μM quercetin or 45 μM isoliquiritigenin for 48 h and subjected to Cignal finder reporter assay. Each reporter is a luciferase construct in which a transcriptional factor specific to a signaling pathway was cloned. Signaling pathways affected by either quercetin or isoliquiritigenin were chosen when their fold changes were immensely higher or lower than negative and positive reporter signals (shaded by red box). Reporters induced by either quercetin or isoliquiritigenin were in blue box and reporters suppressed by either quercetin or isoliquiritigenin were in green box. ERE, HSR, SRE, AP1 reporters were upregulated by quercetin, and these reporters are specific to estrogen pathway, heat shock response pathway, MAPK/ERK pathway and MARK/JNK pathway, respectively. GRE, MTF1, GAS, LXR, SMAD reporters were downregulated by quercetin, and these reporters are specific to glucocorticoid pathway, heavy metal stress pathway, interferon gamma pathway, liver X receptor pathway and TGF-beta pathway, respectively. AARE, SRE, AP1, PPAR, RXR reporters were upregulated by isoliquiritigenin, and these reporters are specific to amino acid deprivation response pathway, MAPK/ERK pathway, MAPK/JNK pathway, PPAR pathway and retinoid X receptor pathway, respectively. IFR1 reporter was downregulated by isoliquiritigenin and this reporter is specific to interferon regulatory factor 1 pathway.

Techniques Used: Transduction, Reporter Assay, Luciferase, Construct, Clone Assay

3) Product Images from "Matrine derivate MASM uncovers a novel function for ribosomal protein S5 in osteoclastogenesis and postmenopausal osteoporosis"

Article Title: Matrine derivate MASM uncovers a novel function for ribosomal protein S5 in osteoclastogenesis and postmenopausal osteoporosis

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.394

M19 inhibits osteoclastogenesis at early stage and inhibits expressions of osteoclastogenesis-related markers. ( a ) Effect of M19 on RANKL-induced primary osteoclast precursor differentiation at different stage. ( b ) Effect of M19 on RANKL-induced RAW264.7 cell differentiation at different stages. ( c ) Western blot and optical density analysis of expression of Trap, Cathepsin K, TRAF 6, MMP9 and CTR with Beta actin as reference. ①RAW264.7 cells; ② RAW264.7 cells induced with M-CSF, RANKL and PBS; ③ RAW264.7 cells induced with M-CSF, RANKL and treated with 1 μ M M19; ④ RAW264.7 cells induced with M-CSF, RANKL and treated with 2 μ M M19; ⑤RAW264.7 cells induced with M-CSF, RANKL and treated with 5 μ M M19 (* P
Figure Legend Snippet: M19 inhibits osteoclastogenesis at early stage and inhibits expressions of osteoclastogenesis-related markers. ( a ) Effect of M19 on RANKL-induced primary osteoclast precursor differentiation at different stage. ( b ) Effect of M19 on RANKL-induced RAW264.7 cell differentiation at different stages. ( c ) Western blot and optical density analysis of expression of Trap, Cathepsin K, TRAF 6, MMP9 and CTR with Beta actin as reference. ①RAW264.7 cells; ② RAW264.7 cells induced with M-CSF, RANKL and PBS; ③ RAW264.7 cells induced with M-CSF, RANKL and treated with 1 μ M M19; ④ RAW264.7 cells induced with M-CSF, RANKL and treated with 2 μ M M19; ⑤RAW264.7 cells induced with M-CSF, RANKL and treated with 5 μ M M19 (* P

Techniques Used: Cell Differentiation, Western Blot, Expressing

4) Product Images from "Protein Arginine Methyltransferase 6 Regulates Embryonic Stem Cell Identity"

Article Title: Protein Arginine Methyltransferase 6 Regulates Embryonic Stem Cell Identity

Journal: Stem Cells and Development

doi: 10.1089/scd.2011.0330

Prmt6-overexpressing cells undergo differentiation. (A) Immunoblot with cell extracts from HM1 cells treated with leukemia inhibitory factor (LIF) removal and retinoic acid (RA) for different days. Antibodies against Prmt6, histone H3K4me3 and H3R2me2 were used. Beta-actin and histone H3 served as loading controls. (B) Prmt6-overexpressing cells lost the expression marker of undifferentiated embryonic stem ( ES) cells, alkaline phosphatase (AP; scale bar=100 μm), compared to the empty vector-transfected cells. Cells were counted using cell counter and result showed that Prmt6-overexpressing cells were 57.3% less than control cells. (C) Immunoblot with chromatin extracts from parental HM1 cells and Prmt6-overexpressing cells using antibodies against Prmt6, histone H3 marks (H3K4 and H3R2) or pluripotency markers (Nanog and Oct4). Beta-actin and histone H3 served as loading controls. (D) Overexpression of Prmt6 induced the high expression levels of certain lineage marker genes, especially endoderm and mesoderm markers. Mean levels expressed relative to the vector control and normalized to beta-actin
Figure Legend Snippet: Prmt6-overexpressing cells undergo differentiation. (A) Immunoblot with cell extracts from HM1 cells treated with leukemia inhibitory factor (LIF) removal and retinoic acid (RA) for different days. Antibodies against Prmt6, histone H3K4me3 and H3R2me2 were used. Beta-actin and histone H3 served as loading controls. (B) Prmt6-overexpressing cells lost the expression marker of undifferentiated embryonic stem ( ES) cells, alkaline phosphatase (AP; scale bar=100 μm), compared to the empty vector-transfected cells. Cells were counted using cell counter and result showed that Prmt6-overexpressing cells were 57.3% less than control cells. (C) Immunoblot with chromatin extracts from parental HM1 cells and Prmt6-overexpressing cells using antibodies against Prmt6, histone H3 marks (H3K4 and H3R2) or pluripotency markers (Nanog and Oct4). Beta-actin and histone H3 served as loading controls. (D) Overexpression of Prmt6 induced the high expression levels of certain lineage marker genes, especially endoderm and mesoderm markers. Mean levels expressed relative to the vector control and normalized to beta-actin

Techniques Used: Expressing, Marker, Plasmid Preparation, Transfection, Over Expression

Prmt6 RNAi results in ES cell differentiation. (A) Depletion of Prmt6 increases the expression level of certain lineage markers, such as Fgf5 . Mean levels are expressed relative to the vector control and normalized to beta-actin expression levels. (B) Immunoblot with chromatin extracts from GFP RNAi cells and Prmt6 RNAi ES cells using antibodies against Prmt6, histone H3 marks (H3K4me3 and H3R2me2), or pluripotency markers (Nanog and Oct4). Beta-actin and histone H3 served as loading controls. (C) Prmt6
Figure Legend Snippet: Prmt6 RNAi results in ES cell differentiation. (A) Depletion of Prmt6 increases the expression level of certain lineage markers, such as Fgf5 . Mean levels are expressed relative to the vector control and normalized to beta-actin expression levels. (B) Immunoblot with chromatin extracts from GFP RNAi cells and Prmt6 RNAi ES cells using antibodies against Prmt6, histone H3 marks (H3K4me3 and H3R2me2), or pluripotency markers (Nanog and Oct4). Beta-actin and histone H3 served as loading controls. (C) Prmt6

Techniques Used: Cell Differentiation, Expressing, Plasmid Preparation

5) Product Images from "STAT3 and HIF1? cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells"

Article Title: STAT3 and HIF1? cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells

Journal: Oncogene

doi: 10.1038/onc.2013.115

Hypoxia activates STAT3 activity in breast cancer cell line MDA-MB-231. ( a ) Western blot analysis of total and phosphorylated (Y705) STAT3 protein levels in normoxic and hypoxic (Hx) RCC4 (pVHL-null), Hep3B and MDA-MB-231 cancer cell lines. Beta-actin
Figure Legend Snippet: Hypoxia activates STAT3 activity in breast cancer cell line MDA-MB-231. ( a ) Western blot analysis of total and phosphorylated (Y705) STAT3 protein levels in normoxic and hypoxic (Hx) RCC4 (pVHL-null), Hep3B and MDA-MB-231 cancer cell lines. Beta-actin

Techniques Used: Activity Assay, Multiple Displacement Amplification, Western Blot

6) Product Images from "Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K‐AKT‐NFATc1 pathway, et al. Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K‐AKT‐NFATc1 pathway"

Article Title: Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K‐AKT‐NFATc1 pathway, et al. Triptolide prevents bone loss via suppressing osteoclastogenesis through inhibiting PI3K‐AKT‐NFATc1 pathway

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.15229

NFATc1 overexpression partially ameliorates the effect of triptolide. A, Formation of tartrate‐resistant acid phosphatase (TRAP)‐positive cells from RAW264.7 cells. B, The resorption area on the bone biomimetic synthetic surface. C, Western blot detected expressions of osteoclastogenesis‐related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP‐9, TRAF6 and TRAP with beta‐actin as reference. D, Expression of phosphorylation of AKT, PI3K and NFATc1 detected by Western blot assay (** P
Figure Legend Snippet: NFATc1 overexpression partially ameliorates the effect of triptolide. A, Formation of tartrate‐resistant acid phosphatase (TRAP)‐positive cells from RAW264.7 cells. B, The resorption area on the bone biomimetic synthetic surface. C, Western blot detected expressions of osteoclastogenesis‐related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP‐9, TRAF6 and TRAP with beta‐actin as reference. D, Expression of phosphorylation of AKT, PI3K and NFATc1 detected by Western blot assay (** P

Techniques Used: Over Expression, Western Blot, Expressing

SC79 partially ameliorate the effect of triptolide. A, Formation of tartrate‐resistant acid phosphatase (TRAP)‐positive cells from RAW264.7 cells. B, The resorption area on the bone biomimetic synthetic surface. C, Western blot detected expressions of osteoclastogenesis‐related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP‐9, TRAF6 and TRAP with beta‐actin as reference. D, Expression of phosphorylation of AKT, PI3K and NFATc1 detected by Western blot assay (** P
Figure Legend Snippet: SC79 partially ameliorate the effect of triptolide. A, Formation of tartrate‐resistant acid phosphatase (TRAP)‐positive cells from RAW264.7 cells. B, The resorption area on the bone biomimetic synthetic surface. C, Western blot detected expressions of osteoclastogenesis‐related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP‐9, TRAF6 and TRAP with beta‐actin as reference. D, Expression of phosphorylation of AKT, PI3K and NFATc1 detected by Western blot assay (** P

Techniques Used: Western Blot, Expressing

Triptolide inhibits expressions of osteoclastogenesis‐related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP‐9, TRAF6 and tartrate‐resistant acid phosphatase with beta‐actin as reference (** P
Figure Legend Snippet: Triptolide inhibits expressions of osteoclastogenesis‐related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP‐9, TRAF6 and tartrate‐resistant acid phosphatase with beta‐actin as reference (** P

Techniques Used: Western Blot, Expressing

7) Product Images from "TACI expression is associated with a mature bone marrow plasma cell signature and C-MAF overexpression in human myeloma cell lines"

Article Title: TACI expression is associated with a mature bone marrow plasma cell signature and C-MAF overexpression in human myeloma cell lines

Journal: Haematologica

doi:

TACI and c-maf expressions are correlated in HMCL (A) Correlation between TACI and c-maf expressions in TACI + HMCL using Affymetrix microarrays or real time RT-PCR. (B) Expression level of c-maf , cyclin D2 and integrin beta7 in TACI + and TACI − HMCL using Affymetrix microarrays. (C) Expression level of TACI and c-maf in HMCL using Affymetrix microarrays, western blot and flow cytometry. For each cell line, the ratios of c-maf and beta actin proteins were determined in order to compare c-maf protein expression between cell lines.
Figure Legend Snippet: TACI and c-maf expressions are correlated in HMCL (A) Correlation between TACI and c-maf expressions in TACI + HMCL using Affymetrix microarrays or real time RT-PCR. (B) Expression level of c-maf , cyclin D2 and integrin beta7 in TACI + and TACI − HMCL using Affymetrix microarrays. (C) Expression level of TACI and c-maf in HMCL using Affymetrix microarrays, western blot and flow cytometry. For each cell line, the ratios of c-maf and beta actin proteins were determined in order to compare c-maf protein expression between cell lines.

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

8) Product Images from "Torin2 targets dysregulated pathways in anaplastic thyroid cancer and inhibits tumor growth and metastasis"

Article Title: Torin2 targets dysregulated pathways in anaplastic thyroid cancer and inhibits tumor growth and metastasis

Journal: Oncotarget

doi:

Effect of Torin2 on mTOR and mTOR-related protein expression and phosphorylation A. Western blot analysis of AKT, phospho-AKT Ser473 , mTOR Ser2448 (mTORC1 site) and total mTOR. ATC cells were treated with Torin2 for 48 hours at T1 = 0.05 μM and T2 = 0.14 μM. Beta-actin was used as a loading control for the mTOR blot because of the higher molecular weight. B. Western blot analysis of downstream targets of mTOR: phospho-S6K (p-S6K), total 70S6K, phospho-4E-BP1 (p-4E-BP1), total 4E-BP1, phospho PRAS40 (p-PRAS40) and total PRAS40 with Torin2 treatment for 48 hours in ATC cells at T1 = 0.05 μM and T2 = 0.14 μM.
Figure Legend Snippet: Effect of Torin2 on mTOR and mTOR-related protein expression and phosphorylation A. Western blot analysis of AKT, phospho-AKT Ser473 , mTOR Ser2448 (mTORC1 site) and total mTOR. ATC cells were treated with Torin2 for 48 hours at T1 = 0.05 μM and T2 = 0.14 μM. Beta-actin was used as a loading control for the mTOR blot because of the higher molecular weight. B. Western blot analysis of downstream targets of mTOR: phospho-S6K (p-S6K), total 70S6K, phospho-4E-BP1 (p-4E-BP1), total 4E-BP1, phospho PRAS40 (p-PRAS40) and total PRAS40 with Torin2 treatment for 48 hours in ATC cells at T1 = 0.05 μM and T2 = 0.14 μM.

Techniques Used: Expressing, Western Blot, Molecular Weight

9) Product Images from "Tissue-specific disallowance of housekeeping genes: The other face of cell differentiation"

Article Title: Tissue-specific disallowance of housekeeping genes: The other face of cell differentiation

Journal: Genome Research

doi: 10.1101/gr.109173.110

Allowed and disallowed genes. ( A ) Microarray expression profiles of pyruvate carboxylase ( Pcx ) versus lactate dehydrogenase A ( Ldha ), which are, respectively, highly expressed and deeply repressed in adult mouse pancreatic beta-cells. Mean mRNA expression
Figure Legend Snippet: Allowed and disallowed genes. ( A ) Microarray expression profiles of pyruvate carboxylase ( Pcx ) versus lactate dehydrogenase A ( Ldha ), which are, respectively, highly expressed and deeply repressed in adult mouse pancreatic beta-cells. Mean mRNA expression

Techniques Used: Microarray, Expressing

10) Product Images from "STAT3 or USF2 Contributes to HIF Target Gene Specificity"

Article Title: STAT3 or USF2 Contributes to HIF Target Gene Specificity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072358

STAT3 or USF2 alone or with HIF1α or HIF2α to activate the cloned promoters of HIF1 or HIF2 target genes in 293T cells. A ) Schematic presentation of the promoters of HIF1 target genes PGK1 and CA9 . B ) Schematic presentation of the promoters/enhancers of HIF2 target genes PAI1 and EPO . Predicted STAT3 {TT(N) 4-6 AA} binding sites (black solid boxes), USF binding sites ( CANNTG ) (gray boxes), and HIF binding sites (HBS, ACGTG) (white boxes) are indicated. Previously validated HIF and USF2 binding sites are indicated by bold boxes. C ) Western blot analysis of Flag-tagged STAT3C, USF2, HIF1αTM and HIF2αTM to monitor the expression of these plasmids in reporter gene assays for Figure 1D-E. Anti-beta actin was for loading control of total protein for this figure and others in the study. D ) Fold of induction of CA9/Luc and PGK1/Luc reporters activated by the indicated plasmids. E ) Fold of induction of PAI1/Luc and EPO/Luc reporters activated by the indicated plasmids.
Figure Legend Snippet: STAT3 or USF2 alone or with HIF1α or HIF2α to activate the cloned promoters of HIF1 or HIF2 target genes in 293T cells. A ) Schematic presentation of the promoters of HIF1 target genes PGK1 and CA9 . B ) Schematic presentation of the promoters/enhancers of HIF2 target genes PAI1 and EPO . Predicted STAT3 {TT(N) 4-6 AA} binding sites (black solid boxes), USF binding sites ( CANNTG ) (gray boxes), and HIF binding sites (HBS, ACGTG) (white boxes) are indicated. Previously validated HIF and USF2 binding sites are indicated by bold boxes. C ) Western blot analysis of Flag-tagged STAT3C, USF2, HIF1αTM and HIF2αTM to monitor the expression of these plasmids in reporter gene assays for Figure 1D-E. Anti-beta actin was for loading control of total protein for this figure and others in the study. D ) Fold of induction of CA9/Luc and PGK1/Luc reporters activated by the indicated plasmids. E ) Fold of induction of PAI1/Luc and EPO/Luc reporters activated by the indicated plasmids.

Techniques Used: Clone Assay, Binding Assay, Western Blot, Expressing

11) Product Images from "Fibroid explants reveal a higher sensitivity against MDM2-inhibitor nutlin-3 than matching myometrium"

Article Title: Fibroid explants reveal a higher sensitivity against MDM2-inhibitor nutlin-3 than matching myometrium

Journal: BMC Women's Health

doi: 10.1186/1472-6874-12-2

Western Blot analyses of explants treated with nutlin-3 reveal a concentration dependent increase of the amount of p53 . A: Western Blot analysis of p53 of explants from an UL (case 700-1) treated with 30 μM and 50 μM nutlin-3 for 72 h shows a concentration-dependent increase of the amount of p53. Lane 1: marker SeeBlue Plus2 Pre-Stained Standard (Invitrogen, Karlsruhe, Germany), lane 2: control without nultin-3, lane 3: 30 μM nutlin-3, lane 4: 50 μM nutlin-3 (left to right). B: p53 protein expression determined after immunoblotting (c.f. A) by ImageJ (as described in the materials and methods section) against beta-actin. Control was set 100%. Ordinate: % change of p53 expression compared to control.
Figure Legend Snippet: Western Blot analyses of explants treated with nutlin-3 reveal a concentration dependent increase of the amount of p53 . A: Western Blot analysis of p53 of explants from an UL (case 700-1) treated with 30 μM and 50 μM nutlin-3 for 72 h shows a concentration-dependent increase of the amount of p53. Lane 1: marker SeeBlue Plus2 Pre-Stained Standard (Invitrogen, Karlsruhe, Germany), lane 2: control without nultin-3, lane 3: 30 μM nutlin-3, lane 4: 50 μM nutlin-3 (left to right). B: p53 protein expression determined after immunoblotting (c.f. A) by ImageJ (as described in the materials and methods section) against beta-actin. Control was set 100%. Ordinate: % change of p53 expression compared to control.

Techniques Used: Western Blot, Concentration Assay, Marker, Staining, Expressing

12) Product Images from "Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation"

Article Title: Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation

Journal: Scientific Reports

doi: 10.1038/srep36338

Characterization of EVs and PK-treated EVs. ( a ) Western blot analysis with EV markers, CD81 and TSG101, in 12 fractions from OptiPrep density gradient. ( b ) The particle number in each OptiPrep fraction was analyzed by nanoparticle tracking analysis. ( c ) Cryo-EM images of non-treated and PK-treated EVs. ( d ) Western blot analysis of non-treated and PK-treated EVs with CD81 and beta-actin.
Figure Legend Snippet: Characterization of EVs and PK-treated EVs. ( a ) Western blot analysis with EV markers, CD81 and TSG101, in 12 fractions from OptiPrep density gradient. ( b ) The particle number in each OptiPrep fraction was analyzed by nanoparticle tracking analysis. ( c ) Cryo-EM images of non-treated and PK-treated EVs. ( d ) Western blot analysis of non-treated and PK-treated EVs with CD81 and beta-actin.

Techniques Used: Western Blot

Validation of surface-accessible proteome and inside-out proteins. ( a ) Western blot analysis of surface-accessible and EV proteome. STUB1, GAPDH, Histone H1, and PCNA are surface-accessible proteome, whereas Flotilin 1 and TSG101 are EV proteome. Relative band intensity was measured. ( b ) Flow cytometry of inside-out membrane proteins. EVs were captured by SCAMP3 (left panel) or STX4 (right panel) antibody conjugated beads and then detected with CD63 or CD81. ( c ) The percentage of SCAMP3, STX4, and beta-actin positive EVs were calculated after incubation with or without 0.1% Tween-20.
Figure Legend Snippet: Validation of surface-accessible proteome and inside-out proteins. ( a ) Western blot analysis of surface-accessible and EV proteome. STUB1, GAPDH, Histone H1, and PCNA are surface-accessible proteome, whereas Flotilin 1 and TSG101 are EV proteome. Relative band intensity was measured. ( b ) Flow cytometry of inside-out membrane proteins. EVs were captured by SCAMP3 (left panel) or STX4 (right panel) antibody conjugated beads and then detected with CD63 or CD81. ( c ) The percentage of SCAMP3, STX4, and beta-actin positive EVs were calculated after incubation with or without 0.1% Tween-20.

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Incubation

13) Product Images from "MiD51 Is Important for Maintaining Mitochondrial Health in Pancreatic Islet and MIN6 Cells"

Article Title: MiD51 Is Important for Maintaining Mitochondrial Health in Pancreatic Islet and MIN6 Cells

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2020.00232

MiD51 expression in MIN6 and primary beta cells. Endogenous MiD51 protein expression is demonstrated in MIN6 ( A , left), primary mouse islet ( B , left) and primary human islet ( C , left) cells with by immunofluorescence. In addition, staining of primary human islet cells with insulin and MiD51 antibodies is shown (D) . Representative images from three independent experiments are shown. Scale bar: 10 μm. Endogenous MiD51 gene expression is shown in MIN6 ( A , right) and primary mouse islet ( B , right) cells after incubation with 5.5 (white bars) and 25 mmol/l glucose (black bars), and in human islet cells after incubation with 11 mmol/l glucose (black bar) ( C , right) for 48 h.
Figure Legend Snippet: MiD51 expression in MIN6 and primary beta cells. Endogenous MiD51 protein expression is demonstrated in MIN6 ( A , left), primary mouse islet ( B , left) and primary human islet ( C , left) cells with by immunofluorescence. In addition, staining of primary human islet cells with insulin and MiD51 antibodies is shown (D) . Representative images from three independent experiments are shown. Scale bar: 10 μm. Endogenous MiD51 gene expression is shown in MIN6 ( A , right) and primary mouse islet ( B , right) cells after incubation with 5.5 (white bars) and 25 mmol/l glucose (black bars), and in human islet cells after incubation with 11 mmol/l glucose (black bar) ( C , right) for 48 h.

Techniques Used: Expressing, Immunofluorescence, Staining, Incubation

14) Product Images from "Knockout of Density-Enhanced Phosphatase-1 Impairs Cerebrovascular Reserve Capacity in an Arteriogenesis Model in Mice"

Article Title: Knockout of Density-Enhanced Phosphatase-1 Impairs Cerebrovascular Reserve Capacity in an Arteriogenesis Model in Mice

Journal: BioMed Research International

doi: 10.1155/2013/802149

(a) Endothelial cells were transfected with DEP-1 siRNA or nontargeting siRNA for 48 hours. Representative microphotographs were taken 48 hours after transfection. Protein lysates were WGA precipitated and immunoblotted against DEP-1. Beta-actin immunoblots served as loading control. Representative qPCR analyses of DEP-1 (b) and PDGF-B (c) expressions in endothelial cells 48 hours after DEP-1 siRNA knockdown are depicted ( n = 3).
Figure Legend Snippet: (a) Endothelial cells were transfected with DEP-1 siRNA or nontargeting siRNA for 48 hours. Representative microphotographs were taken 48 hours after transfection. Protein lysates were WGA precipitated and immunoblotted against DEP-1. Beta-actin immunoblots served as loading control. Representative qPCR analyses of DEP-1 (b) and PDGF-B (c) expressions in endothelial cells 48 hours after DEP-1 siRNA knockdown are depicted ( n = 3).

Techniques Used: Transfection, Whole Genome Amplification, Western Blot, Real-time Polymerase Chain Reaction

15) Product Images from "EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival"

Article Title: EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004979

Expression of miR-BARTs in AGS cells suppressed protein expression from 10 candidate pro-apoptotic genes. (A) Representative Western blots showing the level of protein expression for 10 candidate target genes in AGS cells stably expressing the indicated miR-BART pre-miRNA compared to control AGS cells (Neg). Relative expression levels of each candidate gene product were normalized to an endogenous beta-actin control. Note that only CASZ1a expression was significantly inhibited by pre-miR-BART3, while CASZ1b expression was unaffected. Where more than one protein band was observed, the relevant band is indicated by an arrow. (B) Relative protein expression levels of candidate pro-apoptotic target genes in AGS cells stably expressing the indicated miR-BART pre-miRNA, compared to control AGS cells (Neg; normalized to 1). Average of four independent experiments with SD indicated. * Students’ T-Test, p
Figure Legend Snippet: Expression of miR-BARTs in AGS cells suppressed protein expression from 10 candidate pro-apoptotic genes. (A) Representative Western blots showing the level of protein expression for 10 candidate target genes in AGS cells stably expressing the indicated miR-BART pre-miRNA compared to control AGS cells (Neg). Relative expression levels of each candidate gene product were normalized to an endogenous beta-actin control. Note that only CASZ1a expression was significantly inhibited by pre-miR-BART3, while CASZ1b expression was unaffected. Where more than one protein band was observed, the relevant band is indicated by an arrow. (B) Relative protein expression levels of candidate pro-apoptotic target genes in AGS cells stably expressing the indicated miR-BART pre-miRNA, compared to control AGS cells (Neg; normalized to 1). Average of four independent experiments with SD indicated. * Students’ T-Test, p

Techniques Used: Expressing, Western Blot, Stable Transfection

16) Product Images from "The Human Papillomavirus 16 E6 Protein Can Either Protect or Further Sensitize Cells to TNF: Effect of Dose (HPV E6 Sensitizes and Protects Cells from TNF)"

Article Title: The Human Papillomavirus 16 E6 Protein Can Either Protect or Further Sensitize Cells to TNF: Effect of Dose (HPV E6 Sensitizes and Protects Cells from TNF)

Journal: Cell death and differentiation

doi: 10.1038/sj.cdd.4401678

Transfected HA-E6 retains biological activity in both sensitive and resistant clones. A) ELISA measurement of baseline and induced levels of p53 in E6-expressing clones. The p53 ELISA was used to measure the amount of p53 in the indicated clones both before and after a 4 hr treatment with the genotoxin MNNG (10 μg/ml). Error bars represent the standard deviation. B) Immunoblot measurement of baseline and induced levels of p53 in U2OS and U2OSE64a cells. U2OS and U2OSE64a cells were treated with the genotoxin mitomycin C for the indicated times. Lysates were prepared, subjected to SDS-PAGE and transferred to a membrane, then immunoblotted with antibodies directed against p53 (DO-7) (top panel) or anti-beta actin (bottom panel).
Figure Legend Snippet: Transfected HA-E6 retains biological activity in both sensitive and resistant clones. A) ELISA measurement of baseline and induced levels of p53 in E6-expressing clones. The p53 ELISA was used to measure the amount of p53 in the indicated clones both before and after a 4 hr treatment with the genotoxin MNNG (10 μg/ml). Error bars represent the standard deviation. B) Immunoblot measurement of baseline and induced levels of p53 in U2OS and U2OSE64a cells. U2OS and U2OSE64a cells were treated with the genotoxin mitomycin C for the indicated times. Lysates were prepared, subjected to SDS-PAGE and transferred to a membrane, then immunoblotted with antibodies directed against p53 (DO-7) (top panel) or anti-beta actin (bottom panel).

Techniques Used: Transfection, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Clone Assay, Standard Deviation, SDS Page

17) Product Images from "A Matrine Derivative M54 Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Targeting Ribosomal Protein S5"

Article Title: A Matrine Derivative M54 Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Targeting Ribosomal Protein S5

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00022

M54 inhibits expressions of osteoclastogenesis-related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP-9, NFATc1, and TRAP with Beta actin as reference. ➀RAW264.7 cells; ➁RAW264.7 cells induced with M-CSF, RANKL and PBS; ➂RAW264.7 cells induced with M-CSF, RANKL and treated with 1 μM M54; ➃RAW264.7 cells induced with M-CSF, RANKL and treated with 2 μM M54; ➄RAW264.7 cells induced with M-CSF, RANKL and treated with 4 μM M54. The membranes were cut according to the molecular size of the target protein and reincubated with primary antibodies but using the same loading control ( ∗∗ P
Figure Legend Snippet: M54 inhibits expressions of osteoclastogenesis-related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP-9, NFATc1, and TRAP with Beta actin as reference. ➀RAW264.7 cells; ➁RAW264.7 cells induced with M-CSF, RANKL and PBS; ➂RAW264.7 cells induced with M-CSF, RANKL and treated with 1 μM M54; ➃RAW264.7 cells induced with M-CSF, RANKL and treated with 2 μM M54; ➄RAW264.7 cells induced with M-CSF, RANKL and treated with 4 μM M54. The membranes were cut according to the molecular size of the target protein and reincubated with primary antibodies but using the same loading control ( ∗∗ P

Techniques Used: Western Blot, Expressing

RPS5 regulates expressions of osteoclastogenesis-related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP-9, NFATc1, and TRAP with Beta actin as reference. ➀RAW264.7 cells; ➁RAW264.7 induced with M-CSF, RANKL and PBS; ➂RAW264.7 cells infected with Lv-shRNA-RPS5 and induced with M-CSF, RANKL and PBS; ➃RAW264.7 cells induced with M-CSF, RANKL and treated with 4 μM M54; ➄RAW264.7 cells infected with Lv-shRNA-RPS5, induced with M-CSF, RANKL and treated with 4 μM M54. The membranes were cut according to the molecular size of the target protein and reincubated with primary antibodies but using the same loading control ( ∗∗ P
Figure Legend Snippet: RPS5 regulates expressions of osteoclastogenesis-related markers. Western blot and optical density analysis of expression of Cathepsin K, CTR, MMP-9, NFATc1, and TRAP with Beta actin as reference. ➀RAW264.7 cells; ➁RAW264.7 induced with M-CSF, RANKL and PBS; ➂RAW264.7 cells infected with Lv-shRNA-RPS5 and induced with M-CSF, RANKL and PBS; ➃RAW264.7 cells induced with M-CSF, RANKL and treated with 4 μM M54; ➄RAW264.7 cells infected with Lv-shRNA-RPS5, induced with M-CSF, RANKL and treated with 4 μM M54. The membranes were cut according to the molecular size of the target protein and reincubated with primary antibodies but using the same loading control ( ∗∗ P

Techniques Used: Western Blot, Expressing, Infection, shRNA

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Article Title: High-Frequency Repetitive Magnetic Stimulation Enhances the Expression of Brain-Derived Neurotrophic Factor Through Activation of Ca2+–Calmodulin-Dependent Protein Kinase II–cAMP-Response Element-Binding Protein Pathway
Article Snippet: .. Membranes were blocked and then incubated overnight at 4°C with anti-CaMKII (1:1,000 dilution, Abcam), anti-p-CREB (1:1,000 dilution, Santa Cruz Biotechnology), anti-BDNF (1:1,000 dilution, Abcam), and anti-ACTIN (1:5,000 dilution, Santacruz) antibodies. .. The next day, blots were washed three times with 1× TBS plus Tween 20 (Biosesang, Sungnam, Korea) and incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:4,000 dilution, Santa Cruz).

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Article Title: Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells
Article Snippet: Antibodies Antibodies used: anti-core protein monoclonal antibody (Affinity BioReagents, Inc., Golden, CO); anti-NS3, anti-FAK, anti-paxillin, anti-alpha-actinin, anti-alpha-smooth muscle actin, anti-beta-actin and anti-phosphotyrosine monoclonal antibodies (Santa Cruz Biotech.); peroxidase-conjugated goat anti rabbit and anti-mouse IgG (Sigma-Aldrich Inc.); FITC-conjugated anti-mouse and TRIC-conjugated anti-rabbit (Sigma-Aldrich Inc.).

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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 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti ß actin
    PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or <t>ß-actin</t> was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p
    Anti ß Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti β actin antibody
    The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to <t>β-actin</t> in 0% FBS group was set to 1. *** p
    Anti β Actin Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 707 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    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

    PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p

    Journal: Scientific Reports

    Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity

    doi: 10.1038/s41598-019-40238-w

    Figure Lengend Snippet: PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p

    Article Snippet: Antibodies The following antibodies were used in the present study: anti-δ-catenin (#611537, BD Bioscience, San Jose, CA, USA), anti-GFP (#G1544, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tubulin (#T9026, Sigma-Aldrich, St Louis, MO, USA), anti-lamin B (SC-6216, Santa Cruz Biotechnology), anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF (SC-13124, Santa Cruz Biotechnology), anti-E-cadherin (BD Bioscience), anti-acetylated-Lys (Cell Signaling, Beverly, MA, USA), anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) (Cell Signaling), anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 (Calbiochem, San Diego, CA, USA).

    Techniques: Activity Assay, Inhibition, Transfection, Incubation

    Multiple lysine residues in the N-terminus are responsible for PCAF-mediated δ-catenin downregulation. ( A ) Schematic representation of the triple arginine mutation at Lys360, Lys371, and Lys428 (FL KR), and the deletion/arginine mutation constructs of δ-catenin 1–499, 1–499 KR, 85–499, 85–499 KR, 1–499∆N KR, and 325–499 KR. ( B ) Deletion mutants 85–499 KR and 325–499 KR of δ-catenin were not affected by PCAF. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibody. ( C ) 3-MA restored the downregulated FL KR, 85–499, and 1–499∆N KR mutants of δ-catenin except the 85–499 KR mutation. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs and incubated with 3-MA (1 mM) for 24 h, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibodies. ( D ) PCAF did not acetylate δ-catenin 85–499 KR mutation. HEK293T cells were transfected with full length GFP-δ-catenin or 85–499 KR mutant together with or without Flag-PCAF, and each cell lysates were subjected to immunoprecipitation with anti-acetylated-lysine, followed by immunoblotting of precipitated proteins. α-Tubulin or ß-actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p

    Journal: Scientific Reports

    Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity

    doi: 10.1038/s41598-019-40238-w

    Figure Lengend Snippet: Multiple lysine residues in the N-terminus are responsible for PCAF-mediated δ-catenin downregulation. ( A ) Schematic representation of the triple arginine mutation at Lys360, Lys371, and Lys428 (FL KR), and the deletion/arginine mutation constructs of δ-catenin 1–499, 1–499 KR, 85–499, 85–499 KR, 1–499∆N KR, and 325–499 KR. ( B ) Deletion mutants 85–499 KR and 325–499 KR of δ-catenin were not affected by PCAF. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibody. ( C ) 3-MA restored the downregulated FL KR, 85–499, and 1–499∆N KR mutants of δ-catenin except the 85–499 KR mutation. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs and incubated with 3-MA (1 mM) for 24 h, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibodies. ( D ) PCAF did not acetylate δ-catenin 85–499 KR mutation. HEK293T cells were transfected with full length GFP-δ-catenin or 85–499 KR mutant together with or without Flag-PCAF, and each cell lysates were subjected to immunoprecipitation with anti-acetylated-lysine, followed by immunoblotting of precipitated proteins. α-Tubulin or ß-actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p

    Article Snippet: Antibodies The following antibodies were used in the present study: anti-δ-catenin (#611537, BD Bioscience, San Jose, CA, USA), anti-GFP (#G1544, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tubulin (#T9026, Sigma-Aldrich, St Louis, MO, USA), anti-lamin B (SC-6216, Santa Cruz Biotechnology), anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF (SC-13124, Santa Cruz Biotechnology), anti-E-cadherin (BD Bioscience), anti-acetylated-Lys (Cell Signaling, Beverly, MA, USA), anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) (Cell Signaling), anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 (Calbiochem, San Diego, CA, USA).

    Techniques: Mutagenesis, Construct, Transfection, Expressing, Incubation, Immunoprecipitation

    PCAF and HDACs regulate the acetylation status and levels of δ-catenin. ( A , B ) PCAF decreases δ-catenin levels. HEK293T ( A ) and δ-catenin-overexpressing CWR22Rv-1 (Rv/δ) ( B ) cells were transfected as indicated, and cell lysates were subjected to immunoblotting. ( C – F ) PCAF interacts with and acetylates δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoprecipitation with anti-δ-catenin ( C ), anti-Flag ( D ), or anti-acetylated-lysine ( E , F ), followed by immunoblotting of precipitated proteins. ( G – I ) HDACs deacetylate and upregulate δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoblotting ( G ) or immunoprecipitation with anti-acetylated-lysine ( H ). At 12 h post-transfection of HEK293T cells with GFP-δ-catenin and HDAC1, cells were treated with 0.2 µM trichostatin A (TSA) or transfected with Flag-PCAF, and incubated for 24 h, followed by immunoblotting with anti-δ-catenin and anti-Flag antibodies ( I ). α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin/HDACs ratios from three different experimental results are shown as a bar graph ( G ). Values are presented as the mean ± SEM. “−”, Mock transfection or vehicle treatment.

    Journal: Scientific Reports

    Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity

    doi: 10.1038/s41598-019-40238-w

    Figure Lengend Snippet: PCAF and HDACs regulate the acetylation status and levels of δ-catenin. ( A , B ) PCAF decreases δ-catenin levels. HEK293T ( A ) and δ-catenin-overexpressing CWR22Rv-1 (Rv/δ) ( B ) cells were transfected as indicated, and cell lysates were subjected to immunoblotting. ( C – F ) PCAF interacts with and acetylates δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoprecipitation with anti-δ-catenin ( C ), anti-Flag ( D ), or anti-acetylated-lysine ( E , F ), followed by immunoblotting of precipitated proteins. ( G – I ) HDACs deacetylate and upregulate δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoblotting ( G ) or immunoprecipitation with anti-acetylated-lysine ( H ). At 12 h post-transfection of HEK293T cells with GFP-δ-catenin and HDAC1, cells were treated with 0.2 µM trichostatin A (TSA) or transfected with Flag-PCAF, and incubated for 24 h, followed by immunoblotting with anti-δ-catenin and anti-Flag antibodies ( I ). α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin/HDACs ratios from three different experimental results are shown as a bar graph ( G ). Values are presented as the mean ± SEM. “−”, Mock transfection or vehicle treatment.

    Article Snippet: Antibodies The following antibodies were used in the present study: anti-δ-catenin (#611537, BD Bioscience, San Jose, CA, USA), anti-GFP (#G1544, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tubulin (#T9026, Sigma-Aldrich, St Louis, MO, USA), anti-lamin B (SC-6216, Santa Cruz Biotechnology), anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF (SC-13124, Santa Cruz Biotechnology), anti-E-cadherin (BD Bioscience), anti-acetylated-Lys (Cell Signaling, Beverly, MA, USA), anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) (Cell Signaling), anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 (Calbiochem, San Diego, CA, USA).

    Techniques: Transfection, Immunoprecipitation, Incubation

    The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy

    doi: 10.3390/ijms19123903

    Figure Lengend Snippet: The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Article Snippet: Goat antibodies directed against LDLR (C-7, sc-18823), HRP-labeled anti-goat IgG (sc-2354) and anti-β-actin antibody (sc-8432) were from Santa Cruz Biotechnology (Heidelberg, German).

    Techniques: Expressing, Cell Culture, Concentration Assay, Western Blot

    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