Structured Review

Millipore antibody against β actin
Effect of apoE depletion on eNOS expression in HUVEC. Panel A. HDL2 isolated from the homozygous carrier of the R37X CETP mutation were separated by 2D electrophoresis, followed by anti apoA-I or anti apoE immunodetection, before (−) and after (+) incubation with heparin-MnCl 2 . Panel B. Cells were incubated overnight with HDL2 (1 mg/ml) from the homozygous carrier of the R37X CETP mutation and from controls (n = 3) before (full bars) and after (open bars) treatment with heparin-MnCl 2 . Western blot analysis of eNOS protein was performed, and eNOS protein band intensities were normalized for <t>β-actin</t> values and expressed as fold of increase in treated vs. untreated.
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1) Product Images from "eNOS Activation by HDL Is Impaired in Genetic CETP Deficiency"

Article Title: eNOS Activation by HDL Is Impaired in Genetic CETP Deficiency

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095925

Effect of apoE depletion on eNOS expression in HUVEC. Panel A. HDL2 isolated from the homozygous carrier of the R37X CETP mutation were separated by 2D electrophoresis, followed by anti apoA-I or anti apoE immunodetection, before (−) and after (+) incubation with heparin-MnCl 2 . Panel B. Cells were incubated overnight with HDL2 (1 mg/ml) from the homozygous carrier of the R37X CETP mutation and from controls (n = 3) before (full bars) and after (open bars) treatment with heparin-MnCl 2 . Western blot analysis of eNOS protein was performed, and eNOS protein band intensities were normalized for β-actin values and expressed as fold of increase in treated vs. untreated.
Figure Legend Snippet: Effect of apoE depletion on eNOS expression in HUVEC. Panel A. HDL2 isolated from the homozygous carrier of the R37X CETP mutation were separated by 2D electrophoresis, followed by anti apoA-I or anti apoE immunodetection, before (−) and after (+) incubation with heparin-MnCl 2 . Panel B. Cells were incubated overnight with HDL2 (1 mg/ml) from the homozygous carrier of the R37X CETP mutation and from controls (n = 3) before (full bars) and after (open bars) treatment with heparin-MnCl 2 . Western blot analysis of eNOS protein was performed, and eNOS protein band intensities were normalized for β-actin values and expressed as fold of increase in treated vs. untreated.

Techniques Used: Expressing, Isolation, Mutagenesis, Two-Dimensional Gel Electrophoresis, Immunodetection, Incubation, Western Blot

2) Product Images from "MS-5, a Naphthalene Derivative, Induces the Apoptosis of an Ovarian Cancer Cell CAOV-3 by Interfering with the Reactive Oxygen Species Generation"

Article Title: MS-5, a Naphthalene Derivative, Induces the Apoptosis of an Ovarian Cancer Cell CAOV-3 by Interfering with the Reactive Oxygen Species Generation

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2018.020

Effect of MS-5 on cellular markers of apoptosis in CAOV-3 cells. Cells were treated with indicated concentrations of MS-5 for 24 h. (A) Western blot analysis of cell lysates for cleaved caspase-3, -7, and -9, cleaved PARP, cytochrome c, Bcl-2, Bax, and Survivin. β-actin was used as a loading control and Cox4 was used as a loading control for mitochondrial fraction. (B) The cytoplasmic level of caspase-3 was assayed by Western blotting after separation of cytosolic and mitochondrial fractions.
Figure Legend Snippet: Effect of MS-5 on cellular markers of apoptosis in CAOV-3 cells. Cells were treated with indicated concentrations of MS-5 for 24 h. (A) Western blot analysis of cell lysates for cleaved caspase-3, -7, and -9, cleaved PARP, cytochrome c, Bcl-2, Bax, and Survivin. β-actin was used as a loading control and Cox4 was used as a loading control for mitochondrial fraction. (B) The cytoplasmic level of caspase-3 was assayed by Western blotting after separation of cytosolic and mitochondrial fractions.

Techniques Used: Mass Spectrometry, Western Blot

Cell cycle arrest induced by MS-5 treatment. (A) The cell cycle distribution was analyzed by flow cytometry after PI staining. (B) Cells were treated with indicated concentrations of MS-5 for 24 h. Western blot analysis of cleaved CDK2, cyclin D1, cyclin E, p16, p21, and p27 protein levels. β-actin was used as a loading control.
Figure Legend Snippet: Cell cycle arrest induced by MS-5 treatment. (A) The cell cycle distribution was analyzed by flow cytometry after PI staining. (B) Cells were treated with indicated concentrations of MS-5 for 24 h. Western blot analysis of cleaved CDK2, cyclin D1, cyclin E, p16, p21, and p27 protein levels. β-actin was used as a loading control.

Techniques Used: Mass Spectrometry, Flow Cytometry, Cytometry, Staining, Western Blot

3) Product Images from "Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3"

Article Title: Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3

Journal: Oncogene

doi: 10.1038/onc.2017.164

Stat3 gets recruited to the promoters of Oct4, Nanog and Sox2 and honokiol inhibits stemness transcription factors via inhibiting Stat3. ( a ) Breast cancer cells were treated with 5 μM Honokiol (HNK) for indicated time-intervals. Total protein lysates were immunoblotted for phospho-Stat3 and total Stat3 expression. β-actin was used as a control. ( b ) MCF7 cells were transfected with Stat3-overexpression (Stat3 O/E) followed by 5 μM Honokiol (HNK) treatment and co-treatment with HNK and Stattic as indicated. Total RNA was analyzed for the expression of Oct4, Nanog and Sox2. Bar graph shows fold change in gene expression. * P
Figure Legend Snippet: Stat3 gets recruited to the promoters of Oct4, Nanog and Sox2 and honokiol inhibits stemness transcription factors via inhibiting Stat3. ( a ) Breast cancer cells were treated with 5 μM Honokiol (HNK) for indicated time-intervals. Total protein lysates were immunoblotted for phospho-Stat3 and total Stat3 expression. β-actin was used as a control. ( b ) MCF7 cells were transfected with Stat3-overexpression (Stat3 O/E) followed by 5 μM Honokiol (HNK) treatment and co-treatment with HNK and Stattic as indicated. Total RNA was analyzed for the expression of Oct4, Nanog and Sox2. Bar graph shows fold change in gene expression. * P

Techniques Used: Expressing, Transfection, Over Expression

LKB1-AMPK axis plays an important role in honokiol-mediated inhibition of stemness transcription factors. ( a ) LKB1-depleted (LKB1 shRNA 1–2 ) and vector control (pLKO.1) MDA-MB-231 cells were treated with 5 μM honokiol (HNK). Total RNA was examined for the expression of Oct4, Nanog and Sox2 as indicted. β-actin was used as control. ( b ) Total protein lysates of LKB1-depleted (LKB1 shRNA 1–2 ) and vector control (pLKO.1) breast cancer cells treated with 5 μM honokiol (HNK) were analyzed for the expression of Nanog and Sox2 as indicated. β-actin was used as loading-control. ( c ) LKB1-depleted (LKB1 shRNA1–2 ) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1 O/E ); total protein lysates were examined for LKB1 expression as indicated using immunoblot analysis. ( d ) LKB1-depleted (LKB1 shRNA1 ) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1 O/E ) followed by treatment with 5 μM honokiol (HNK). Total protein lysates were examined for Oct4 and Nanog. β-actin was used as loading-control. ( e ) Bar diagram shows quantitation of western blot signals. * P
Figure Legend Snippet: LKB1-AMPK axis plays an important role in honokiol-mediated inhibition of stemness transcription factors. ( a ) LKB1-depleted (LKB1 shRNA 1–2 ) and vector control (pLKO.1) MDA-MB-231 cells were treated with 5 μM honokiol (HNK). Total RNA was examined for the expression of Oct4, Nanog and Sox2 as indicted. β-actin was used as control. ( b ) Total protein lysates of LKB1-depleted (LKB1 shRNA 1–2 ) and vector control (pLKO.1) breast cancer cells treated with 5 μM honokiol (HNK) were analyzed for the expression of Nanog and Sox2 as indicated. β-actin was used as loading-control. ( c ) LKB1-depleted (LKB1 shRNA1–2 ) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1 O/E ); total protein lysates were examined for LKB1 expression as indicated using immunoblot analysis. ( d ) LKB1-depleted (LKB1 shRNA1 ) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1 O/E ) followed by treatment with 5 μM honokiol (HNK). Total protein lysates were examined for Oct4 and Nanog. β-actin was used as loading-control. ( e ) Bar diagram shows quantitation of western blot signals. * P

Techniques Used: Inhibition, shRNA, Plasmid Preparation, Multiple Displacement Amplification, Expressing, Transfection, Quantitation Assay, Western Blot

4) Product Images from "Differential Role of PTEN in Transforming Growth Factor β (TGF-β) Effects on Proliferation and Migration in Prostate Cancer Cells"

Article Title: Differential Role of PTEN in Transforming Growth Factor β (TGF-β) Effects on Proliferation and Migration in Prostate Cancer Cells

Journal: The Prostate

doi: 10.1002/pros.23482

TGF-β induces phosphorylation of Smad proteins in DU145 and RWPE1 cells (Western blot analyses of phosphorylated Smad2 (pSmad2) and Smad3 (pSmad3) in ( A ) DU145 cells and ( B ) RWPE1 cells after treatment with TGF-β1 or TGF-β3 (5ng/ml) for 15 and 30 minutes. Total Smad (Smad2/3) and β-actin were used as loading controls. Each bar represents mean ± SEM (n=3). Different letters denote significant differences among various groups ( P
Figure Legend Snippet: TGF-β induces phosphorylation of Smad proteins in DU145 and RWPE1 cells (Western blot analyses of phosphorylated Smad2 (pSmad2) and Smad3 (pSmad3) in ( A ) DU145 cells and ( B ) RWPE1 cells after treatment with TGF-β1 or TGF-β3 (5ng/ml) for 15 and 30 minutes. Total Smad (Smad2/3) and β-actin were used as loading controls. Each bar represents mean ± SEM (n=3). Different letters denote significant differences among various groups ( P

Techniques Used: Western Blot

Basal expression of PTEN and the levels of pAKT Ser473 in prostate cell lines ( A ) RT-PCR was performed using total RNA from RWPE1, LNCaP, DU145, and PC3 cells to determine mRNA levels of PTEN. L-19 served as a loading control and was used to normalize mRNA levels in all cell line samples. No reverse transcriptase (RT) samples derived from the same RNAs were also included. ( B ) Levels of PTEN protein and pAKT Ser473 as determined by western blotting analysis. β-actin and total AKT (tAKT) were used as loading controls. ( C ) Western blot analysis was performed to determine relative protein levels of PTEN and pAKT Ser473 in DU145 cells after transfection with control siRNA or PTEN siRNA. Total AKT (tAKT) and β-actin were used as loading controls.
Figure Legend Snippet: Basal expression of PTEN and the levels of pAKT Ser473 in prostate cell lines ( A ) RT-PCR was performed using total RNA from RWPE1, LNCaP, DU145, and PC3 cells to determine mRNA levels of PTEN. L-19 served as a loading control and was used to normalize mRNA levels in all cell line samples. No reverse transcriptase (RT) samples derived from the same RNAs were also included. ( B ) Levels of PTEN protein and pAKT Ser473 as determined by western blotting analysis. β-actin and total AKT (tAKT) were used as loading controls. ( C ) Western blot analysis was performed to determine relative protein levels of PTEN and pAKT Ser473 in DU145 cells after transfection with control siRNA or PTEN siRNA. Total AKT (tAKT) and β-actin were used as loading controls.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Western Blot, Transfection

5) Product Images from "NF-κB Regulates Caspase-4 Expression and Sensitizes Neuroblastoma Cells to Fas-Induced Apoptosis"

Article Title: NF-κB Regulates Caspase-4 Expression and Sensitizes Neuroblastoma Cells to Fas-Induced Apoptosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117953

Caspase-4 acts upstream of PARP and caspase-3, but may play downstream of caspase-8. ( A ) SH-EP1 cells transfected with control vector (Ctl) or DN-Cas4 expression vector (DN-Cas4) were treated with Fas antibody (100 ng/ml) for 0, 4, 8, or 16 h, and Western blotting was performed with antibodies against cleaved PARP and caspase-3. β-actin was used as a loading control. Results are representative of at least three experiments. ( B ) In the presence or absence of caspase-8 inhibitor, z-LETD-fmk (30 μM), Fas-induced cell death was examined using crystal violet staining. * P
Figure Legend Snippet: Caspase-4 acts upstream of PARP and caspase-3, but may play downstream of caspase-8. ( A ) SH-EP1 cells transfected with control vector (Ctl) or DN-Cas4 expression vector (DN-Cas4) were treated with Fas antibody (100 ng/ml) for 0, 4, 8, or 16 h, and Western blotting was performed with antibodies against cleaved PARP and caspase-3. β-actin was used as a loading control. Results are representative of at least three experiments. ( B ) In the presence or absence of caspase-8 inhibitor, z-LETD-fmk (30 μM), Fas-induced cell death was examined using crystal violet staining. * P

Techniques Used: Transfection, Plasmid Preparation, CTL Assay, Expressing, Western Blot, Staining

NF-κB inhibition protects neuroblastoma cells from Fas-induced apoptosis. ( A ) SH-EP1 cells transfected with control vector (Ctl) or DN-IκBα expression vector (DN-IκBα) were treated with anti-Fas antibody (100 ng/ml) for 2 h. NF-κB activation was analyzed using NF-κB p65 reporter assay. ( B ) Cells were treated with anti-Fas antibody of different concentrations for 1 d, and cell viability was examined using crystal violet staining. ( C ) Fas-treated cells were lyzed and Western blotting was performed with antibodies against cleaved PARP and caspase-8. Membranes were re-probed with β-actin as a loading control. Results are representative of at least three experiments. ** P
Figure Legend Snippet: NF-κB inhibition protects neuroblastoma cells from Fas-induced apoptosis. ( A ) SH-EP1 cells transfected with control vector (Ctl) or DN-IκBα expression vector (DN-IκBα) were treated with anti-Fas antibody (100 ng/ml) for 2 h. NF-κB activation was analyzed using NF-κB p65 reporter assay. ( B ) Cells were treated with anti-Fas antibody of different concentrations for 1 d, and cell viability was examined using crystal violet staining. ( C ) Fas-treated cells were lyzed and Western blotting was performed with antibodies against cleaved PARP and caspase-8. Membranes were re-probed with β-actin as a loading control. Results are representative of at least three experiments. ** P

Techniques Used: Inhibition, Transfection, Plasmid Preparation, CTL Assay, Expressing, Activation Assay, Reporter Assay, Staining, Western Blot

NF-κB is involved in Fas-induced cell apoptosis in neuroblastoma cells. ( A ) SH-EP1 cells were treated with agonistic anti-Fas antibody (100 ng/ml) for 1 d, and photographed live. Pictures are shown at ×100 magnification. ( B ) Immunocytochemistry targeting NF-κB p65 (green) was performed at 1 h after Fas treatment. Arrows indicate cells with p65 nuclear translocation. ( C ) Treated cells were lysed and Western blotting was performed with antibody against the cleaved PARP. Membranes were re-probed with β-actin as a loading control. Results are representative of at least three experiments. ( D ) After Fas treatment, NF-κB activation was analyzed using NF-κB p65 reporter assay. Data are from three repeated experiments and shown as average ± s.e.m. (bars) values. ** P
Figure Legend Snippet: NF-κB is involved in Fas-induced cell apoptosis in neuroblastoma cells. ( A ) SH-EP1 cells were treated with agonistic anti-Fas antibody (100 ng/ml) for 1 d, and photographed live. Pictures are shown at ×100 magnification. ( B ) Immunocytochemistry targeting NF-κB p65 (green) was performed at 1 h after Fas treatment. Arrows indicate cells with p65 nuclear translocation. ( C ) Treated cells were lysed and Western blotting was performed with antibody against the cleaved PARP. Membranes were re-probed with β-actin as a loading control. Results are representative of at least three experiments. ( D ) After Fas treatment, NF-κB activation was analyzed using NF-κB p65 reporter assay. Data are from three repeated experiments and shown as average ± s.e.m. (bars) values. ** P

Techniques Used: Immunocytochemistry, Translocation Assay, Western Blot, Activation Assay, Reporter Assay

Caspase-4 is required for Fas-induced neuroblastoma cell apoptosis. ( A ) SH-EP1 cells transfected with control vector (Ctl) or DN-Cas4 expression vector (DN-Cas4) were subjected to Western blotting analysis with anti-caspase-4 antibody. #1, #7, and #10 represent selected single cell clones with different levels of overexpression of DN-Cas4.Membranes were re-probed with β-actin as a loading control. Results are representative of three experiments. ( B ) DN-Cas4 cells were treated with anti-Fas antibody of different concentrations for 1 d, and cell viability was examined using crystal violet staining. * P
Figure Legend Snippet: Caspase-4 is required for Fas-induced neuroblastoma cell apoptosis. ( A ) SH-EP1 cells transfected with control vector (Ctl) or DN-Cas4 expression vector (DN-Cas4) were subjected to Western blotting analysis with anti-caspase-4 antibody. #1, #7, and #10 represent selected single cell clones with different levels of overexpression of DN-Cas4.Membranes were re-probed with β-actin as a loading control. Results are representative of three experiments. ( B ) DN-Cas4 cells were treated with anti-Fas antibody of different concentrations for 1 d, and cell viability was examined using crystal violet staining. * P

Techniques Used: Transfection, Plasmid Preparation, CTL Assay, Expressing, Western Blot, Clone Assay, Over Expression, Staining

6) Product Images from "Endothelial Differentiation by Multipotent Fetal Mouse Lung Mesenchymal Cells"

Article Title: Endothelial Differentiation by Multipotent Fetal Mouse Lung Mesenchymal Cells

Journal: Stem Cells and Development

doi: 10.1089/scd.2011.0219

Endothelial growth media-2 (EGM-2) stimulates vascular endothelial differentiation of fetal lung mesenchymal cells. (A) Immunoblot for the SV40 Large T antigen shows degradation over time after culture at 37°C. β-actin included as loading
Figure Legend Snippet: Endothelial growth media-2 (EGM-2) stimulates vascular endothelial differentiation of fetal lung mesenchymal cells. (A) Immunoblot for the SV40 Large T antigen shows degradation over time after culture at 37°C. β-actin included as loading

Techniques Used:

7) Product Images from "Activating transcription factor 3 promotes malignance of lung cancer cells in vitro"

Article Title: Activating transcription factor 3 promotes malignance of lung cancer cells in vitro

Journal: Thoracic Cancer

doi: 10.1111/1759-7714.12421

Activating transcription factor 3 ( ATF 3) expression was upregulated in non‐small cell lung cancer ( NSCLC) tissues. ( a ) Determination of ATF 3 messenger RNA (m RNA ) level by quantitative real‐time PCR . Δ CtN : threshold cycle ( C t) value of β‐actin was subtracted from the C t value of ATF 3 of paired normal tissue. Δ CtT : C t value of β‐actin was subtracted from that of ATF 3 of NSCLC tissue. Bar value (Δ CtN − Δ CtT ) represents the difference between ATF 3 m RNA level in NSCLC and paired normal tissues. Bar value ≤−1 indicates that ATF 3 expression was decreased in NSCLC tissues. Bar value ≥1 indicates that ATF 3 expression of was increased in NSCLC tissues. ( b ) Determination of protein level of ATF 3 by immunoblotting. The lower panel shows the quantitative results of ATF 3 protein in the paired samples. ( c ) Immunohistochemical staining of paired samples with ATF 3 antibody.
Figure Legend Snippet: Activating transcription factor 3 ( ATF 3) expression was upregulated in non‐small cell lung cancer ( NSCLC) tissues. ( a ) Determination of ATF 3 messenger RNA (m RNA ) level by quantitative real‐time PCR . Δ CtN : threshold cycle ( C t) value of β‐actin was subtracted from the C t value of ATF 3 of paired normal tissue. Δ CtT : C t value of β‐actin was subtracted from that of ATF 3 of NSCLC tissue. Bar value (Δ CtN − Δ CtT ) represents the difference between ATF 3 m RNA level in NSCLC and paired normal tissues. Bar value ≤−1 indicates that ATF 3 expression was decreased in NSCLC tissues. Bar value ≥1 indicates that ATF 3 expression of was increased in NSCLC tissues. ( b ) Determination of protein level of ATF 3 by immunoblotting. The lower panel shows the quantitative results of ATF 3 protein in the paired samples. ( c ) Immunohistochemical staining of paired samples with ATF 3 antibody.

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

8) Product Images from "Tid1-S regulates the mitochondrial localization of EGFR in non-small cell lung carcinoma"

Article Title: Tid1-S regulates the mitochondrial localization of EGFR in non-small cell lung carcinoma

Journal: Oncogenesis

doi: 10.1038/oncsis.2017.62

Effects of Tid1-S overexpression or knockdown on the translocation of EGFR into mitochondria. ( a ) Schematic illustration of wild-type Tid1-S-HA (Tid1-S-Wt) and the DnaJ domain mutant Tid1-S-HA (Tid1-S-Mut), in which the amino acid sequence H121P122D123 (wild-type) was replaced with Q121N122A123 (mutant) in the DnaJ domain of Tid1-S. ( b ) CL1-5 cells were transfected with Tid1-S-Wt, Tid1-S-Mut, or empty vector (control) and cultured for 24 h. Representative IF staining with anti-HA, -EGFR and -MTCOI antibodies is shown in the left panel. The colocalization of EGFR and MTCO1 in mitochondria could be visualized as yellow-color in the EGFR/MTCO1 Merge. The colocalization of HA, EGFR, and MTCOI in mitochondria was detected as white color in the HA/EGFR/MTCO1 Merge. The expression of HA-tagged Tid1-S-Wt or Tid1-S-Mut in the transfected CL1-5 cells was determined by Western blotting, and is shown in the right-upper panel. The arrows indicated by ‘up-S’ and ‘p-S’ show the positions of the unprocessed form (up) and the processed form (p) of Tid1-S-HA (S), respectively. β-Actin was used as a loading control. The colocalization of HA-Tid1-S, EGFR and MTCOI in~200 cells was analyzed using the MetaMorph software. The scores are expressed as a fold-increase over that of the vector-transfected control cells, which was set as 1. The data shown in the right-lower panel were from three independent experiments. ( c ) A549 cells were transfected with siTid1 or siN for 72 h. Representative IF staining with anti-EGFR and anti-MTCOI antibodies is shown in the left panel. The expression level of Tid1 in the transfected cells was analyzed by Western blotting, and is shown in the middle panel. β-Actin was used as a loading control. The scores for the colocalization of EGFR and MTCOI in~200 cells was analyzed using the MetaMorph software (right panel). The data shown are the means±SD from three independent experiments; * P
Figure Legend Snippet: Effects of Tid1-S overexpression or knockdown on the translocation of EGFR into mitochondria. ( a ) Schematic illustration of wild-type Tid1-S-HA (Tid1-S-Wt) and the DnaJ domain mutant Tid1-S-HA (Tid1-S-Mut), in which the amino acid sequence H121P122D123 (wild-type) was replaced with Q121N122A123 (mutant) in the DnaJ domain of Tid1-S. ( b ) CL1-5 cells were transfected with Tid1-S-Wt, Tid1-S-Mut, or empty vector (control) and cultured for 24 h. Representative IF staining with anti-HA, -EGFR and -MTCOI antibodies is shown in the left panel. The colocalization of EGFR and MTCO1 in mitochondria could be visualized as yellow-color in the EGFR/MTCO1 Merge. The colocalization of HA, EGFR, and MTCOI in mitochondria was detected as white color in the HA/EGFR/MTCO1 Merge. The expression of HA-tagged Tid1-S-Wt or Tid1-S-Mut in the transfected CL1-5 cells was determined by Western blotting, and is shown in the right-upper panel. The arrows indicated by ‘up-S’ and ‘p-S’ show the positions of the unprocessed form (up) and the processed form (p) of Tid1-S-HA (S), respectively. β-Actin was used as a loading control. The colocalization of HA-Tid1-S, EGFR and MTCOI in~200 cells was analyzed using the MetaMorph software. The scores are expressed as a fold-increase over that of the vector-transfected control cells, which was set as 1. The data shown in the right-lower panel were from three independent experiments. ( c ) A549 cells were transfected with siTid1 or siN for 72 h. Representative IF staining with anti-EGFR and anti-MTCOI antibodies is shown in the left panel. The expression level of Tid1 in the transfected cells was analyzed by Western blotting, and is shown in the middle panel. β-Actin was used as a loading control. The scores for the colocalization of EGFR and MTCOI in~200 cells was analyzed using the MetaMorph software (right panel). The data shown are the means±SD from three independent experiments; * P

Techniques Used: Over Expression, Translocation Assay, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Cell Culture, Staining, Expressing, Western Blot, Software

9) Product Images from "Discovery of an Indirubin Derivative as a Novel c-Met Kinase Inhibitor with In Vitro Anti-Tumor Effects"

Article Title: Discovery of an Indirubin Derivative as a Novel c-Met Kinase Inhibitor with In Vitro Anti-Tumor Effects

Journal: Biomolecules & Therapeutics

doi: 10.4062/biomolther.2018.091

Inhibitory effects of LDD-1937 on c-Met autophosphorylation and the signal transduction pathway in SNU-638 cells. (A) Cells treated with the indicated concentrations of LDD-1937 for 5 h were subjected to immunofluorescence staining using c-Met and phosphorylated c-Met antibodies (red) and DAPI (blue) (scale bars, 50 μm). Representative images from three independent experiments are shown. (B) Cells were incubated in the absence or presence of increasing concentrations of LDD-1937 for 5 h. Western blot using specific antibodies was performed as described. β-Actin western blot was used as a loading control.
Figure Legend Snippet: Inhibitory effects of LDD-1937 on c-Met autophosphorylation and the signal transduction pathway in SNU-638 cells. (A) Cells treated with the indicated concentrations of LDD-1937 for 5 h were subjected to immunofluorescence staining using c-Met and phosphorylated c-Met antibodies (red) and DAPI (blue) (scale bars, 50 μm). Representative images from three independent experiments are shown. (B) Cells were incubated in the absence or presence of increasing concentrations of LDD-1937 for 5 h. Western blot using specific antibodies was performed as described. β-Actin western blot was used as a loading control.

Techniques Used: Transduction, Immunofluorescence, Staining, Incubation, Western Blot

10) Product Images from "Endothelin-1 exposure on postnatal day 7 alters expression of the endothelin B receptor and behavioral sensitivity to endothelin-1 on postnatal day 11"

Article Title: Endothelin-1 exposure on postnatal day 7 alters expression of the endothelin B receptor and behavioral sensitivity to endothelin-1 on postnatal day 11

Journal: Neuroscience letters

doi: 10.1016/j.neulet.2008.12.027

Sex-dependent endothelin-1 induced changes in ET B  receptor expression in the skin. A. In male rats, hind paw intraplantar administration of ET-1, but not saline, on P7 decreases expression of the ET B  receptor in the ipsilateral plantar hind paw skin on P11. In female rats, hind paw intraplantar administration of ET-1, but not saline, on P7 increases expression of the ET B  receptor in the ipsilateral plantar hind paw skin on P11. B. Densitometry analysis of ET B  receptor expression in the ipsilateral left plantar hindpaw skin. ET B  receptor expression is normalized to β actin and expressed at a percentage of expression in skin from same sex normal naïve P11 rats. *,**P
Figure Legend Snippet: Sex-dependent endothelin-1 induced changes in ET B receptor expression in the skin. A. In male rats, hind paw intraplantar administration of ET-1, but not saline, on P7 decreases expression of the ET B receptor in the ipsilateral plantar hind paw skin on P11. In female rats, hind paw intraplantar administration of ET-1, but not saline, on P7 increases expression of the ET B receptor in the ipsilateral plantar hind paw skin on P11. B. Densitometry analysis of ET B receptor expression in the ipsilateral left plantar hindpaw skin. ET B receptor expression is normalized to β actin and expressed at a percentage of expression in skin from same sex normal naïve P11 rats. *,**P

Techniques Used: Expressing

11) Product Images from "Garlic Oil Suppressed Nitrosodiethylamine-Induced Hepatocarcinoma in Rats by Inhibiting PI3K-AKT-NF-κB Pathway"

Article Title: Garlic Oil Suppressed Nitrosodiethylamine-Induced Hepatocarcinoma in Rats by Inhibiting PI3K-AKT-NF-κB Pathway

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.10785

Effect of GO and NDEA on total AKT, p-AKT (Thr308), p-AKT (Ser473) and p-AKT (Tyr450) protein contents. A: A representative immunoblot. B: Data presented the expressions of total AKT, p-AKT (Thr 308), p-AKT (Ser473) and p-AKT (Tyr450) as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P
Figure Legend Snippet: Effect of GO and NDEA on total AKT, p-AKT (Thr308), p-AKT (Ser473) and p-AKT (Tyr450) protein contents. A: A representative immunoblot. B: Data presented the expressions of total AKT, p-AKT (Thr 308), p-AKT (Ser473) and p-AKT (Tyr450) as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P

Techniques Used:

Effect of GO and NDEA on the protein expressions of COX-2, iNOS and VEGF. A: A representative immunoblot. B: Data presented the expressions of COX-2, iNOS and VEGF as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P
Figure Legend Snippet: Effect of GO and NDEA on the protein expressions of COX-2, iNOS and VEGF. A: A representative immunoblot. B: Data presented the expressions of COX-2, iNOS and VEGF as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P

Techniques Used:

Effect of GO and NDEA on PI3K-p85 and PI3K-p110 protein contents. A: A representative immunoblot. B: Data presented the expression of p85 and p110 as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P
Figure Legend Snippet: Effect of GO and NDEA on PI3K-p85 and PI3K-p110 protein contents. A: A representative immunoblot. B: Data presented the expression of p85 and p110 as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P

Techniques Used: Expressing

Effect of GO and NDEA on the protein levels of IκB, p-IκB, NF-κB p65 and p-NF-κB p65. A: A representative immunoblot. B: Data presented the expressions of IκB, p-IκB, NF-κB p65 and p-NF-κB p65 as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P
Figure Legend Snippet: Effect of GO and NDEA on the protein levels of IκB, p-IκB, NF-κB p65 and p-NF-κB p65. A: A representative immunoblot. B: Data presented the expressions of IκB, p-IκB, NF-κB p65 and p-NF-κB p65 as percentage of control group (mean ± SD) in triplicate. The protein levels were quantified with β-actin as an internal control. * P

Techniques Used:

Related Articles

Blocking Assay:

Article Title: The Novel Antitubulin Agent TR-764 Strongly Reduces Tumor Vasculature and Inhibits HIF-1α Activation
Article Snippet: The protein concentration was determined using the BCA protein assay reagents (Pierce), equal amounts of protein (10 μg) were resolved by SDS PAGE (7.5–15% acrylamide gels) and transferred to PVDF Hybond-p membrane (GE Healthcare). .. Membranes were blocked with 3% BSA blocking buffer (Sigma-Aldrich) and incubated overnight at 4 °C with primary antibodies against Src Y416 (Cell Signaling), Src (Src-1M-341, Santa Cruz), FAK Y397 (Beckton Dickinson), FAK (C-20, Santa Cruz), RhoA (Santa Cruz), Rock1 (Santa Cruz), Cdc42 (Cell Signaling), β-catenin Y142 (Abcam), β-catenin (Abcam), VE-cadherin Y658 (Abcam), VE-cadherin (D87F2 XP®, Cell Signaling), MLC2 S19 (Cell Signaling), MLC2 (Cell Signaling), HIF-1α (BD Biosciences), HIF-2 (Novus Biologicals), GLUT1 (H-43, Santa Cruz), β-actin (Sigma-Aldrich). .. Membranes were next incubated with peroxidase-labeled secondary antibodies for 60 min. All membranes were visualized using ECL Select (GE Healthcare) and images were acquired by Alliance LD2 scan (UVITEC Cambridge).

Incubation:

Article Title: The Novel Antitubulin Agent TR-764 Strongly Reduces Tumor Vasculature and Inhibits HIF-1α Activation
Article Snippet: The protein concentration was determined using the BCA protein assay reagents (Pierce), equal amounts of protein (10 μg) were resolved by SDS PAGE (7.5–15% acrylamide gels) and transferred to PVDF Hybond-p membrane (GE Healthcare). .. Membranes were blocked with 3% BSA blocking buffer (Sigma-Aldrich) and incubated overnight at 4 °C with primary antibodies against Src Y416 (Cell Signaling), Src (Src-1M-341, Santa Cruz), FAK Y397 (Beckton Dickinson), FAK (C-20, Santa Cruz), RhoA (Santa Cruz), Rock1 (Santa Cruz), Cdc42 (Cell Signaling), β-catenin Y142 (Abcam), β-catenin (Abcam), VE-cadherin Y658 (Abcam), VE-cadherin (D87F2 XP®, Cell Signaling), MLC2 S19 (Cell Signaling), MLC2 (Cell Signaling), HIF-1α (BD Biosciences), HIF-2 (Novus Biologicals), GLUT1 (H-43, Santa Cruz), β-actin (Sigma-Aldrich). .. Membranes were next incubated with peroxidase-labeled secondary antibodies for 60 min. All membranes were visualized using ECL Select (GE Healthcare) and images were acquired by Alliance LD2 scan (UVITEC Cambridge).

Western Blot:

Article Title: IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment
Article Snippet: Real-time RT-PCR for IGFBP3 was done as described previously using β-actin as an internal control [ , ]. .. Western blotting was done as described previously [ , ] using the following primary antibodies at the indicated titers; anti-IGFBP3 (DSL- ; Diagnostic Systems Laboratories, Webster, TX) (1:1000), anti-HIF-1α (610958; BD Biosciences) (1:1000) and anti-β-actin (AC74; Sigma-Aldrich, St. Louis, MO) (1:5000). .. Data from experiments are presented as mean ± standard error (n=3) or mean ± standard deviation (n=8) and were analyzed by two-tailed Student’s t test.

Diagnostic Assay:

Article Title: IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment
Article Snippet: Real-time RT-PCR for IGFBP3 was done as described previously using β-actin as an internal control [ , ]. .. Western blotting was done as described previously [ , ] using the following primary antibodies at the indicated titers; anti-IGFBP3 (DSL- ; Diagnostic Systems Laboratories, Webster, TX) (1:1000), anti-HIF-1α (610958; BD Biosciences) (1:1000) and anti-β-actin (AC74; Sigma-Aldrich, St. Louis, MO) (1:5000). .. Data from experiments are presented as mean ± standard error (n=3) or mean ± standard deviation (n=8) and were analyzed by two-tailed Student’s t test.

FLAG-tag:

Article Title: Virus-induced unfolded protein response attenuates anti-viral defenses via phosphorylation-dependent degradation of the Type I interferon receptor
Article Snippet: .. Antibodies against pSTAT1, p-eIF2α, p-β-catenin, β-catenin, IRE1α (Cell Signaling), STAT1 (Cell Signaling), eIF2α (Biosources), hIFNAR1, PKR, c-Jun, IκBα (Santa Cruz), mIFNAR1 (R & D Systems), Flag tag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol), ISG15 and PERK (Rockland) were used for immunoprecipitation and immunoblotting. ..

Immunoprecipitation:

Article Title: Virus-induced unfolded protein response attenuates anti-viral defenses via phosphorylation-dependent degradation of the Type I interferon receptor
Article Snippet: .. Antibodies against pSTAT1, p-eIF2α, p-β-catenin, β-catenin, IRE1α (Cell Signaling), STAT1 (Cell Signaling), eIF2α (Biosources), hIFNAR1, PKR, c-Jun, IκBα (Santa Cruz), mIFNAR1 (R & D Systems), Flag tag, β-actin (Sigma) and ubiquitin (clone FK2, Biomol), ISG15 and PERK (Rockland) were used for immunoprecipitation and immunoblotting. ..

Protein Extraction:

Article Title: Clinical Characteristics of Connective Tissue Nevi in Tuberous Sclerosis Complex With Special Emphasis on Shagreen Patches
Article Snippet: For Western blot analysis, cells grown from samples of shagreen patch, angiofibroma, ungual fibroma, fibrous cephalic plaque, and normal-appearing skin were seeded overnight into 60-mm dishes at 5 × 105 cells in DMEM (Dulbecco Modified Eagle Medium) with 10% FBS (fetal bovine serum) and switched to serum-free DMEM for 24 hours. .. Cells were lysed in protein extraction buffer and immunoblotting performed as previously described with anti-Tuberin/TSC2 (D93F12), anti-Hamartin/TSC1, anti-phospho-S6 ribosomal protein (Ser-235/236), anti-S6 ribosomal protein antibodies (Cell Signaling), β-actin (Sigma-Aldrich), and horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). .. A paired t test was used to analyze elastin stain score for paired samples of shagreen patch and normal skin.

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  • 97
    Millipore mouse monoclonal antibody against β actin
    (overleaf). Effects of tPA and fibrinolytic products released following lysis of a fibrin clot by PVA- and DMAB-FNPs (loaded with 10 μg of tPA) at 0.5 mg/mL NP concentration in a transwell culture (Clot+NPs in insert, EaRASMCs in wells) on (A, B) MMP-2 synthesis, (C, D) MMP-9 synthesis and (E, F) MMP-2 and -9 activity in EaRASMC cultures, as analyzed with western blots and gel zymography, respectively. Representative images of western blots are shown for (A) MMP-2 and (b) MMP-9, with <t>β-actin</t> bands as loading controls. Fold-change in production of (B) MMP-2 and (D) MMP-9 compared to control cultures without NPs. (E) Representative image of gel zymogram, showing MMP-2 and -9 bands. (F) Fold-change in MMP-2 and -9 activities compared to NP-untreated control EaRASMC cultures. (mean ± SD; n = 3/case for western blots and gel zymograms, * denotes p
    Mouse Monoclonal Antibody Against β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore antibodies against β actin
    Induction of Bim is critical for ATF2-associated apoptotic changes of the mitochondria and apoptosis A. B16F10 cells stably transfected with ATF2-specific shRNA were treated with paclitaxel (100 nM) for 24 h. JC-1 flow cytometry analysis was performed, and representative flow cytometry plots of multiple experiments are shown. B. B16F10 cells stably transfected with scrambled or Bim-specific shRNA were treated with paclitaxel (100 nM) for 24 h. A western blot of the indicated proteins in whole-cell lysates is shown. <t>β-actin</t> was included as a loading control. C. B16F10 cells stably transfected with scrambled or Bim shRNA and Bad shRNA were treated with paclitaxel for 24 h. JC-1 flow cytometry analysis was performed, and representative flow cytometry plots are shown. B16F10 cells D. expressing scrambled or Bim shRNA were subjected to treatment with paclitaxel (100 nM) or ABT-737 (1 μM) for 24 h, and A375 cells E. expressing scrambled or Bim shRNA were subjected to vemurafenib (5 μM) treatment for 24 h. Apoptosis was then measured by Annexin V/PI staining (bars, s.e.m.) ( n = 3). Columns represent the mean percentage of annexin V-positive cells from at least three independent experiments; bars, s.e.m. * p
    Antibodies Against β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β actin
    Lactate production is upregulated in ccRCC primary cell cultures (A) Representative western blot of normal cortex ( n = 2) and ccRCC ( n = 3) primary cell culture, showing LDHA and <t>β-actin</t> proteins. In the graph, the normalized LDHA band intensities of normal cortex ( n = 7) and ccRCC ( n = 19) primary cell cultures are shown. ( B) Lactate quantification performed in cell lysates (cells) and conditioned culture medium (media) of normal cortex ( n = 6) and ccRCC ( n = 7) primary cultures. Lactate concentration values were normalized to cell protein concentration. Data expressed as mean ± SEM; * p
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β actin/product/Millipore
    Average 97 stars, based on 1 article reviews
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    β actin - by Bioz Stars, 2021-03
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    (overleaf). Effects of tPA and fibrinolytic products released following lysis of a fibrin clot by PVA- and DMAB-FNPs (loaded with 10 μg of tPA) at 0.5 mg/mL NP concentration in a transwell culture (Clot+NPs in insert, EaRASMCs in wells) on (A, B) MMP-2 synthesis, (C, D) MMP-9 synthesis and (E, F) MMP-2 and -9 activity in EaRASMC cultures, as analyzed with western blots and gel zymography, respectively. Representative images of western blots are shown for (A) MMP-2 and (b) MMP-9, with β-actin bands as loading controls. Fold-change in production of (B) MMP-2 and (D) MMP-9 compared to control cultures without NPs. (E) Representative image of gel zymogram, showing MMP-2 and -9 bands. (F) Fold-change in MMP-2 and -9 activities compared to NP-untreated control EaRASMC cultures. (mean ± SD; n = 3/case for western blots and gel zymograms, * denotes p

    Journal: Materials science & engineering. C, Materials for biological applications

    Article Title: Fibrinolytic PLGA nanoparticles for slow clot lysis within abdominal aortic aneurysms attenuate proteolytic loss of vascular elastic matrix

    doi: 10.1016/j.msec.2015.09.056

    Figure Lengend Snippet: (overleaf). Effects of tPA and fibrinolytic products released following lysis of a fibrin clot by PVA- and DMAB-FNPs (loaded with 10 μg of tPA) at 0.5 mg/mL NP concentration in a transwell culture (Clot+NPs in insert, EaRASMCs in wells) on (A, B) MMP-2 synthesis, (C, D) MMP-9 synthesis and (E, F) MMP-2 and -9 activity in EaRASMC cultures, as analyzed with western blots and gel zymography, respectively. Representative images of western blots are shown for (A) MMP-2 and (b) MMP-9, with β-actin bands as loading controls. Fold-change in production of (B) MMP-2 and (D) MMP-9 compared to control cultures without NPs. (E) Representative image of gel zymogram, showing MMP-2 and -9 bands. (F) Fold-change in MMP-2 and -9 activities compared to NP-untreated control EaRASMC cultures. (mean ± SD; n = 3/case for western blots and gel zymograms, * denotes p

    Article Snippet: Following electrophoresis, the gels were transferred onto nitrocellulose membranes (iBlot® Western Blotting System, Invitrogen), blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature, and finally immunolabeled for 16 h at 4 °C with a rabbit polyclonal antibody against MMP-2 (1:500 dilution; Abcam, Cambridge, MA) or a rabbit monoclonal antibody against MMP-9 (1:500 dilution; Millipore, Inc.), with a mouse monoclonal antibody against β-actin (1:1000 dilution; Sigma-Aldrich) as the loading control.

    Techniques: Lysis, Concentration Assay, Activity Assay, Western Blot, Zymography

    ATF-3 mediates the recruitment of BRG1 and β-actin to the SLC11A1 promoter. HL-60 cells were transfected alone with Mock (transfection reagent only), control siRNA ( CTR ), or siRNA specific for ATF-3. Six hours after transfection, HL-60 cells were

    Journal: The Journal of Biological Chemistry

    Article Title: Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells *

    doi: 10.1074/jbc.M110.185637

    Figure Lengend Snippet: ATF-3 mediates the recruitment of BRG1 and β-actin to the SLC11A1 promoter. HL-60 cells were transfected alone with Mock (transfection reagent only), control siRNA ( CTR ), or siRNA specific for ATF-3. Six hours after transfection, HL-60 cells were

    Article Snippet: A mouse monoclonal antibody against β-actin and a rabbit polyclonal antibody against Brm were purchased from Sigma and Abcam (Cambridge, MA), respectively.

    Techniques: Transfection

    ATF-3 forms a complex with BRG1 and β-actin in response to PMA. A , nuclear extracts ( NE ) prepared from HL-60 cells left untreated or treated with PMA (10 ng/ml) for 24 and 48 h were subjected to immunoprecipitation ( IP ) with an ATF-3 antibody

    Journal: The Journal of Biological Chemistry

    Article Title: Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells *

    doi: 10.1074/jbc.M110.185637

    Figure Lengend Snippet: ATF-3 forms a complex with BRG1 and β-actin in response to PMA. A , nuclear extracts ( NE ) prepared from HL-60 cells left untreated or treated with PMA (10 ng/ml) for 24 and 48 h were subjected to immunoprecipitation ( IP ) with an ATF-3 antibody

    Article Snippet: A mouse monoclonal antibody against β-actin and a rabbit polyclonal antibody against Brm were purchased from Sigma and Abcam (Cambridge, MA), respectively.

    Techniques: Immunoprecipitation

    ATF-3, BRG1, and β-actin cooperate to activate the SLC11A1 promoter. A , HL-60 cells were transiently transfected with luciferase reporter construct −395Luc alone or in combination with different amounts of expression plasmids for BRG1,

    Journal: The Journal of Biological Chemistry

    Article Title: Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells *

    doi: 10.1074/jbc.M110.185637

    Figure Lengend Snippet: ATF-3, BRG1, and β-actin cooperate to activate the SLC11A1 promoter. A , HL-60 cells were transiently transfected with luciferase reporter construct −395Luc alone or in combination with different amounts of expression plasmids for BRG1,

    Article Snippet: A mouse monoclonal antibody against β-actin and a rabbit polyclonal antibody against Brm were purchased from Sigma and Abcam (Cambridge, MA), respectively.

    Techniques: Transfection, Luciferase, Construct, Expressing

    BRG1 and β-actin are recruited to the AP-1-like site in response to PMA. A , nuclear extracts ( NE ) were prepared from untreated HL-60 cells or cells treated with 10 ng/ml PMA for 24 and 48 h. Proteins that bind to the wild-type ( middle panel ) or

    Journal: The Journal of Biological Chemistry

    Article Title: Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells *

    doi: 10.1074/jbc.M110.185637

    Figure Lengend Snippet: BRG1 and β-actin are recruited to the AP-1-like site in response to PMA. A , nuclear extracts ( NE ) were prepared from untreated HL-60 cells or cells treated with 10 ng/ml PMA for 24 and 48 h. Proteins that bind to the wild-type ( middle panel ) or

    Article Snippet: A mouse monoclonal antibody against β-actin and a rabbit polyclonal antibody against Brm were purchased from Sigma and Abcam (Cambridge, MA), respectively.

    Techniques:

    Induction of Bim is critical for ATF2-associated apoptotic changes of the mitochondria and apoptosis A. B16F10 cells stably transfected with ATF2-specific shRNA were treated with paclitaxel (100 nM) for 24 h. JC-1 flow cytometry analysis was performed, and representative flow cytometry plots of multiple experiments are shown. B. B16F10 cells stably transfected with scrambled or Bim-specific shRNA were treated with paclitaxel (100 nM) for 24 h. A western blot of the indicated proteins in whole-cell lysates is shown. β-actin was included as a loading control. C. B16F10 cells stably transfected with scrambled or Bim shRNA and Bad shRNA were treated with paclitaxel for 24 h. JC-1 flow cytometry analysis was performed, and representative flow cytometry plots are shown. B16F10 cells D. expressing scrambled or Bim shRNA were subjected to treatment with paclitaxel (100 nM) or ABT-737 (1 μM) for 24 h, and A375 cells E. expressing scrambled or Bim shRNA were subjected to vemurafenib (5 μM) treatment for 24 h. Apoptosis was then measured by Annexin V/PI staining (bars, s.e.m.) ( n = 3). Columns represent the mean percentage of annexin V-positive cells from at least three independent experiments; bars, s.e.m. * p

    Journal: Oncotarget

    Article Title: Mitochondrial ATF2 translocation contributes to apoptosis induction and BRAF inhibitor resistance in melanoma through the interaction of Bim with VDAC1

    doi:

    Figure Lengend Snippet: Induction of Bim is critical for ATF2-associated apoptotic changes of the mitochondria and apoptosis A. B16F10 cells stably transfected with ATF2-specific shRNA were treated with paclitaxel (100 nM) for 24 h. JC-1 flow cytometry analysis was performed, and representative flow cytometry plots of multiple experiments are shown. B. B16F10 cells stably transfected with scrambled or Bim-specific shRNA were treated with paclitaxel (100 nM) for 24 h. A western blot of the indicated proteins in whole-cell lysates is shown. β-actin was included as a loading control. C. B16F10 cells stably transfected with scrambled or Bim shRNA and Bad shRNA were treated with paclitaxel for 24 h. JC-1 flow cytometry analysis was performed, and representative flow cytometry plots are shown. B16F10 cells D. expressing scrambled or Bim shRNA were subjected to treatment with paclitaxel (100 nM) or ABT-737 (1 μM) for 24 h, and A375 cells E. expressing scrambled or Bim shRNA were subjected to vemurafenib (5 μM) treatment for 24 h. Apoptosis was then measured by Annexin V/PI staining (bars, s.e.m.) ( n = 3). Columns represent the mean percentage of annexin V-positive cells from at least three independent experiments; bars, s.e.m. * p

    Article Snippet: The blots were reprobed with antibodies against β-actin (Sigma) to ensure equal loading and transfer of proteins.

    Techniques: Stable Transfection, Transfection, shRNA, Flow Cytometry, Cytometry, Western Blot, Expressing, Staining

    Bim triggers mitochondrial VDAC upregulation and dimerization, Cytochrome c release and apoptosis A. B16F10 cells stably transfected with empty vector (EV), ATF2 T52A , or ATF2 shRNA were treated with paclitaxel (100 nM) for 24 h. The cells were then incubated with EGS (250 μM, 15 min), followed by SDS-PAGE and western blot analysis using an anti-VDAC1 antibody. The positions of the molecular weight protein standards are provided. Representative figures of multiple experiments are shown. B. B16F10 cells stably transfected with scrambled or Bim shRNA and Bad shRNA were treated with paclitaxel (100 nM) for 24 h. The cells were then subjected to crosslinking with EGS and western blot analysis using an anti-VDAC1 antibody. C. A375 cells stably transfected with scrambled or Bim shRNA and A375R cells were subjected to vemurafenib (5 μM) treatment for 24 h. The cells were then subjected to crosslinking with EGS and western blot analysis using an anti-VDAC1 antibody. D. B16F10 cells in the presence or absence of DIDS (100 μM, 1 hour) were treated with paclitaxel (100 nM) for 24 h. Western blot analysis of cytochrome c in the cytosolic and mitochondrial fractions was performed. β-actin and COX-IV were used as loading controls. Representative figures of multiple experiments are shown. E. B16F10 cells (left panel) and K1735M2 cells (right panel) stably transfected with empty vector (EV) or ATF2 T52A in the presence or absence of DIDS were further treated with paclitaxel (100 nM) for 24 h and subjected to apoptosis analysis using Annexin V/PI staining. Columns represent the mean percentage of annexin V-positive cells from three independent experiments; bars, s.e.m. *, # p

    Journal: Oncotarget

    Article Title: Mitochondrial ATF2 translocation contributes to apoptosis induction and BRAF inhibitor resistance in melanoma through the interaction of Bim with VDAC1

    doi:

    Figure Lengend Snippet: Bim triggers mitochondrial VDAC upregulation and dimerization, Cytochrome c release and apoptosis A. B16F10 cells stably transfected with empty vector (EV), ATF2 T52A , or ATF2 shRNA were treated with paclitaxel (100 nM) for 24 h. The cells were then incubated with EGS (250 μM, 15 min), followed by SDS-PAGE and western blot analysis using an anti-VDAC1 antibody. The positions of the molecular weight protein standards are provided. Representative figures of multiple experiments are shown. B. B16F10 cells stably transfected with scrambled or Bim shRNA and Bad shRNA were treated with paclitaxel (100 nM) for 24 h. The cells were then subjected to crosslinking with EGS and western blot analysis using an anti-VDAC1 antibody. C. A375 cells stably transfected with scrambled or Bim shRNA and A375R cells were subjected to vemurafenib (5 μM) treatment for 24 h. The cells were then subjected to crosslinking with EGS and western blot analysis using an anti-VDAC1 antibody. D. B16F10 cells in the presence or absence of DIDS (100 μM, 1 hour) were treated with paclitaxel (100 nM) for 24 h. Western blot analysis of cytochrome c in the cytosolic and mitochondrial fractions was performed. β-actin and COX-IV were used as loading controls. Representative figures of multiple experiments are shown. E. B16F10 cells (left panel) and K1735M2 cells (right panel) stably transfected with empty vector (EV) or ATF2 T52A in the presence or absence of DIDS were further treated with paclitaxel (100 nM) for 24 h and subjected to apoptosis analysis using Annexin V/PI staining. Columns represent the mean percentage of annexin V-positive cells from three independent experiments; bars, s.e.m. *, # p

    Article Snippet: The blots were reprobed with antibodies against β-actin (Sigma) to ensure equal loading and transfer of proteins.

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, shRNA, Incubation, SDS Page, Western Blot, Molecular Weight, Staining

    Lactate production is upregulated in ccRCC primary cell cultures (A) Representative western blot of normal cortex ( n = 2) and ccRCC ( n = 3) primary cell culture, showing LDHA and β-actin proteins. In the graph, the normalized LDHA band intensities of normal cortex ( n = 7) and ccRCC ( n = 19) primary cell cultures are shown. ( B) Lactate quantification performed in cell lysates (cells) and conditioned culture medium (media) of normal cortex ( n = 6) and ccRCC ( n = 7) primary cultures. Lactate concentration values were normalized to cell protein concentration. Data expressed as mean ± SEM; * p

    Journal: Oncotarget

    Article Title: The glucose and lipid metabolism reprogramming is grade-dependent in clear cell renal cell carcinoma primary cultures and is targetable to modulate cell viability and proliferation

    doi: 10.18632/oncotarget.23056

    Figure Lengend Snippet: Lactate production is upregulated in ccRCC primary cell cultures (A) Representative western blot of normal cortex ( n = 2) and ccRCC ( n = 3) primary cell culture, showing LDHA and β-actin proteins. In the graph, the normalized LDHA band intensities of normal cortex ( n = 7) and ccRCC ( n = 19) primary cell cultures are shown. ( B) Lactate quantification performed in cell lysates (cells) and conditioned culture medium (media) of normal cortex ( n = 6) and ccRCC ( n = 7) primary cultures. Lactate concentration values were normalized to cell protein concentration. Data expressed as mean ± SEM; * p

    Article Snippet: Protein extraction and western blot analysis Thirty μg of protein lysates obtained from first-confluent primary cell cultures, quantified with BCA microassay (Sigma Aldrich, St. Louis, MO) and separated on NuPage 4 to 12% Bis-Tris pre-cast gels (Thermo Fisher, Waltham, MA) [ ], were submitted to western blotting [ ] with mouse monoclonal antibody against PLIN2 (dilution 1:500; AP125, Progen, Heidelberg, Germany), or rabbit polyclonal antibody against LDHA (dilution 1:1000; Cell Signaling, Boston, MA), or against β-actin (dilution 1:1000; Sigma-Aldrich).

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

    Neutral lipid and glycogen storage is decreased and lactate production increased in high-grade ccRCC primary cultures (A) Representative images of low-grade and high-grade ccRCC tissue sections and matched primary cell cultures after Oil Red O (ORO) and Periodic Acid-Schiff (PAS) staining captured at original magnification of 200× (scale bar: 100 μm). ( B) Glycogen quantification performed in normal cortex ( n = 4), low-grade ( n = 7) and high-grade ( n = 8) ccRCC tissue samples. ( C) Neutral lipid quantification in ORO stained slides of normal cortex ( n = 3), low-grade ( n = 7) and high-grade ( n = 8) ccRCC tissue samples. ORO area was quantified in three to six fields per slides and expressed as percentage of total area analyzed. ( D) Glycogen quantification performed in normal cortex ( n = 3), low-grade ( n = 4) and high-grade ( n = 3) ccRCC primary cultures. ( E) Real-time PCR analysis of PLIN2 expression performed in normal cortex ( n = 15), low-grade ( n = 11) and high-grade ( n = 9) ccRCC primary cultures. Box and whiskers plot corresponds to 1–99 th percentiles (bars), 25–75 th percentiles (box), and median (line in box). ( F) Representative western blot of three different normal cortex, low-grade and high-grade ccRCC primary cell cultures showing the PLIN2 and β-actin proteins. The graph shows the normalized PLIN2 band intensities of normal cortex ( n = 8), low-grade ( n = 8) and high-grade ( n = 7) ccRCC primary cultures. To evidence the difference between normal cortex and all ccRCC cultures, in B-F panels the average of low and high-grade ccRCC data is also reported. ( G) Representative western blot of three different normal cortex, low-grade and high-grade ccRCC primary cell cultures showing the LDHA and β-actin proteins. The graph shows the normalized LDHA band intensities of normal cortex ( n = 7), low-grade ( n = 11) and high-grade ( n = 8) ccRCC primary cultures. ( H) Lactate quantification performed in conditioned culture medium of normal cortex ( n = 5), low- grade ( n = 4) and high-grade ( n = 3) ccRCC primary cultures. Data expressed as mean ± SEM; * p

    Journal: Oncotarget

    Article Title: The glucose and lipid metabolism reprogramming is grade-dependent in clear cell renal cell carcinoma primary cultures and is targetable to modulate cell viability and proliferation

    doi: 10.18632/oncotarget.23056

    Figure Lengend Snippet: Neutral lipid and glycogen storage is decreased and lactate production increased in high-grade ccRCC primary cultures (A) Representative images of low-grade and high-grade ccRCC tissue sections and matched primary cell cultures after Oil Red O (ORO) and Periodic Acid-Schiff (PAS) staining captured at original magnification of 200× (scale bar: 100 μm). ( B) Glycogen quantification performed in normal cortex ( n = 4), low-grade ( n = 7) and high-grade ( n = 8) ccRCC tissue samples. ( C) Neutral lipid quantification in ORO stained slides of normal cortex ( n = 3), low-grade ( n = 7) and high-grade ( n = 8) ccRCC tissue samples. ORO area was quantified in three to six fields per slides and expressed as percentage of total area analyzed. ( D) Glycogen quantification performed in normal cortex ( n = 3), low-grade ( n = 4) and high-grade ( n = 3) ccRCC primary cultures. ( E) Real-time PCR analysis of PLIN2 expression performed in normal cortex ( n = 15), low-grade ( n = 11) and high-grade ( n = 9) ccRCC primary cultures. Box and whiskers plot corresponds to 1–99 th percentiles (bars), 25–75 th percentiles (box), and median (line in box). ( F) Representative western blot of three different normal cortex, low-grade and high-grade ccRCC primary cell cultures showing the PLIN2 and β-actin proteins. The graph shows the normalized PLIN2 band intensities of normal cortex ( n = 8), low-grade ( n = 8) and high-grade ( n = 7) ccRCC primary cultures. To evidence the difference between normal cortex and all ccRCC cultures, in B-F panels the average of low and high-grade ccRCC data is also reported. ( G) Representative western blot of three different normal cortex, low-grade and high-grade ccRCC primary cell cultures showing the LDHA and β-actin proteins. The graph shows the normalized LDHA band intensities of normal cortex ( n = 7), low-grade ( n = 11) and high-grade ( n = 8) ccRCC primary cultures. ( H) Lactate quantification performed in conditioned culture medium of normal cortex ( n = 5), low- grade ( n = 4) and high-grade ( n = 3) ccRCC primary cultures. Data expressed as mean ± SEM; * p

    Article Snippet: Protein extraction and western blot analysis Thirty μg of protein lysates obtained from first-confluent primary cell cultures, quantified with BCA microassay (Sigma Aldrich, St. Louis, MO) and separated on NuPage 4 to 12% Bis-Tris pre-cast gels (Thermo Fisher, Waltham, MA) [ ], were submitted to western blotting [ ] with mouse monoclonal antibody against PLIN2 (dilution 1:500; AP125, Progen, Heidelberg, Germany), or rabbit polyclonal antibody against LDHA (dilution 1:1000; Cell Signaling, Boston, MA), or against β-actin (dilution 1:1000; Sigma-Aldrich).

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