Structured Review

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: 99/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy"

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

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123903

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
Figure Legend 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

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

2) Product Images from "Increased LEF1 Expression and Decreased Notch2 Expression Are Strong Predictors of Poor Outcomes in Colorectal Cancer Patients"

Article Title: Increased LEF1 Expression and Decreased Notch2 Expression Are Strong Predictors of Poor Outcomes in Colorectal Cancer Patients

Journal: Disease markers

doi: 10.1155/2013/983981

Western blot analysis of LEF1 and Notch2 in CRC tissues and paratumorous normal colorectal tissues.  β -actin was used as an internal control. (a) LEF1 expression in CRC tissues and the corresponding paratumorous normal colorectal tissues of the same patient. (b) Notch2 expression in CRC tissues and corresponding paratumorous normal colorectal tissues of the same patient. (c) The percentage of LEF1 and Notch2 positive specimens in CRC tissues (tumor) and corresponding paratumorous normal colorectal tissues (normal).  χ 2  test was used for statistical analyses. ** P
Figure Legend Snippet: Western blot analysis of LEF1 and Notch2 in CRC tissues and paratumorous normal colorectal tissues. β -actin was used as an internal control. (a) LEF1 expression in CRC tissues and the corresponding paratumorous normal colorectal tissues of the same patient. (b) Notch2 expression in CRC tissues and corresponding paratumorous normal colorectal tissues of the same patient. (c) The percentage of LEF1 and Notch2 positive specimens in CRC tissues (tumor) and corresponding paratumorous normal colorectal tissues (normal). χ 2 test was used for statistical analyses. ** P

Techniques Used: Western Blot, Expressing

3) Product Images from "Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice"

Article Title: Ameliorative Effects of Scopoletin from Crossostephium chinensis against Inflammation Pain and Its Mechanisms in Mice

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/595603

Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2.  β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the  average value  for three  individual animals .  ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P
Figure Legend Snippet: Inhibition of iNOS and COX-2 protein expression by scopoletin induced by Carr in mice paw edema for 5th hour. Normal control received 0.9% normal saline. Animals treated with scopoletin (1, 5, and 10 mg/kg) and Indo to injection of Carr right hind paws. The right hind paw tissues were taken at the 5 hour. Then the homogenate was centrifuged and tissue suspended were then prepared and subjected to western blotting using an antibody specific for iNOS and COX-2. β -actin was used as an internal control. (a) Representative western blot from two separate experiments was shown. (b) Relative iNOS and COX-2 protein levels were calculated with reference to Carr-injected mouse. Each point represents the average value for three individual animals . ### compared with sample of control group. The data were presented as mean ± S.E.M. for three different experiments performed in triplicate. ** P

Techniques Used: Inhibition, Expressing, Mouse Assay, Injection, Western Blot

4) Product Images from "Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway"

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

Journal: BMC Research Notes

doi: 10.1186/1756-0500-4-551

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

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

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

Techniques Used: Dominant Negative Mutation, Western Blot, Transfection

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

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

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

Techniques Used: Western Blot, Transfection, Immunoprecipitation, Expressing

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

Techniques Used: Western Blot, Transfection, Stable Transfection

5) Product Images from "hCLOCK Causes Rho-Kinase-Mediated Endothelial Dysfunction and NF-κB-Mediated Inflammatory Responses"

Article Title: hCLOCK Causes Rho-Kinase-Mediated Endothelial Dysfunction and NF-κB-Mediated Inflammatory Responses

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2015/671839

Induction of the RhoA and NF- κ B pathways by hCLOCK. (a) Western blot analysis of RhoA pathway effector ROCK1 and NF- κ B pathway effectors COX-2, IL-6, and p-P65 in HUVECs transduced with control vector or hCLOCK-overexpressing vector. Expression levels are normalized to  β -actin. (b) Western blot analysis showing activated RhoA normalized to total RhoA in HUVECs transduced with control vector or hCLOCK-overexpressing vector. (c) Western blot analysis of the same RhoA and NF- κ B pathway effectors in HUVECs transduced with scrambled control vector or shCLOCK vector. (d) Western blot analysis showing activated RhoA normalized to total RhoA in HUVECs transduced with scrambled control vector or shCLOCK vector.
Figure Legend Snippet: Induction of the RhoA and NF- κ B pathways by hCLOCK. (a) Western blot analysis of RhoA pathway effector ROCK1 and NF- κ B pathway effectors COX-2, IL-6, and p-P65 in HUVECs transduced with control vector or hCLOCK-overexpressing vector. Expression levels are normalized to β -actin. (b) Western blot analysis showing activated RhoA normalized to total RhoA in HUVECs transduced with control vector or hCLOCK-overexpressing vector. (c) Western blot analysis of the same RhoA and NF- κ B pathway effectors in HUVECs transduced with scrambled control vector or shCLOCK vector. (d) Western blot analysis showing activated RhoA normalized to total RhoA in HUVECs transduced with scrambled control vector or shCLOCK vector.

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

Effect of hypoxia over time on protein expression levels of hCLOCK and RhoA in HUVECs. (a) Western blot analysis was performed to evaluate Rac1 and CLOCK protein expression levels in HUVECs exposed to a hypoxic environment. Expression levels are measured at 0, 12, and 24 hours, normalized to  β -actin control. (b) RhoA activation assay and Western blot analysis were performed to measure protein expression levels of activated GTPase-bound RhoA in HUVECs exposed to a hypoxic environment. Expression levels are measured at 0, 12, and 24 hours, normalized to total cellular RhoA.
Figure Legend Snippet: Effect of hypoxia over time on protein expression levels of hCLOCK and RhoA in HUVECs. (a) Western blot analysis was performed to evaluate Rac1 and CLOCK protein expression levels in HUVECs exposed to a hypoxic environment. Expression levels are measured at 0, 12, and 24 hours, normalized to β -actin control. (b) RhoA activation assay and Western blot analysis were performed to measure protein expression levels of activated GTPase-bound RhoA in HUVECs exposed to a hypoxic environment. Expression levels are measured at 0, 12, and 24 hours, normalized to total cellular RhoA.

Techniques Used: Expressing, Western Blot, Activation Assay

6) Product Images from "The effects of Caffeoylserotonin on inhibition of melanogenesis through the downregulation of MITF via the reduction of intracellular cAMP and acceleration of ERK activation in B16 murine melanoma cells"

Article Title: The effects of Caffeoylserotonin on inhibition of melanogenesis through the downregulation of MITF via the reduction of intracellular cAMP and acceleration of ERK activation in B16 murine melanoma cells

Journal: BMB Reports

doi: 10.5483/BMBRep.2012.45.12.039

Effects of CaS on the phosphorylation of ERK in B16F10 cells. (A) Melanocytes were treated with α-MSH and 20 μM CaS for the indicated times. (B) Cells were treated with α-MSH and 20 μM CaS for the indicated times in the presence or absence of PD98059 (50 μM). Whole-cell lysates were then subjected to Western blotting using antibodies against ERK, p-ERK, MITF, and TYR. Equal protein loading was confirmed by reaction with β-actin.
Figure Legend Snippet: Effects of CaS on the phosphorylation of ERK in B16F10 cells. (A) Melanocytes were treated with α-MSH and 20 μM CaS for the indicated times. (B) Cells were treated with α-MSH and 20 μM CaS for the indicated times in the presence or absence of PD98059 (50 μM). Whole-cell lysates were then subjected to Western blotting using antibodies against ERK, p-ERK, MITF, and TYR. Equal protein loading was confirmed by reaction with β-actin.

Techniques Used: Western Blot

Effects of CaS on the expression of melanogenesis-related genes. The B16F10 cells were incubated in the presence of α-MSH and then treated with different concentrations of CaS (1, 5, 10, and 20 μM) for 24 h. (A) Total RNA was extracted and analyzed by RT-PCR for the expression of MITF, TYR, TRP-1, and TRP-2. PCR product levels were measured by densitometry, with the values from CaS-treated cells being quantified relative to the α-MSH-treated control. (B) Intracellular TYR, TRP-2, TRP-1, and MITF levels were measured by Western blotting. Protein levels were quantified by densitometry relative to those of the α-MSH-treated control (after normalisation relative to β-actin, shown just below the gel data). *P < 0.01, **P < 0.001, and ***P < 0.0001 vs. the α-MSH-treated control.
Figure Legend Snippet: Effects of CaS on the expression of melanogenesis-related genes. The B16F10 cells were incubated in the presence of α-MSH and then treated with different concentrations of CaS (1, 5, 10, and 20 μM) for 24 h. (A) Total RNA was extracted and analyzed by RT-PCR for the expression of MITF, TYR, TRP-1, and TRP-2. PCR product levels were measured by densitometry, with the values from CaS-treated cells being quantified relative to the α-MSH-treated control. (B) Intracellular TYR, TRP-2, TRP-1, and MITF levels were measured by Western blotting. Protein levels were quantified by densitometry relative to those of the α-MSH-treated control (after normalisation relative to β-actin, shown just below the gel data). *P < 0.01, **P < 0.001, and ***P < 0.0001 vs. the α-MSH-treated control.

Techniques Used: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot

7) Product Images from "Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma"

Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma

Journal: International Journal of Endocrinology

doi: 10.1155/2015/834137

Brightness was quantified by image analysis software (Image Pro-Plus ver. 7.0) was measured (upper left). Tissue microarray analysis of 60 cases.  β -actin was used as a positive control and normal rabbit IgG as a negative control (Gremlin, FITC; CD34, PI).
Figure Legend Snippet: Brightness was quantified by image analysis software (Image Pro-Plus ver. 7.0) was measured (upper left). Tissue microarray analysis of 60 cases. β -actin was used as a positive control and normal rabbit IgG as a negative control (Gremlin, FITC; CD34, PI).

Techniques Used: Software, Microarray, Positive Control, Negative Control

8) Product Images from "Shotgun proteomics deciphered age/division of labor-related functional specification of three honeybee (Apis mellifera L.) exocrine glands"

Article Title: Shotgun proteomics deciphered age/division of labor-related functional specification of three honeybee (Apis mellifera L.) exocrine glands

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191344

Validation of variable protein expression levels based on immunoblotting analyses. Immunoblotting analysis for MRJP2, aldolase, acetyl-CoA acyltransferase 2, and IDGF4 were performed with two independent biologic samples (red and green bars), and the band intensity for each protein in each gland was normalized to that of beta-actin. The normalized protein expression levels in each gland were compared with the spectral counts (SC) of each protein in each gland normalized to that of beta-actin (blue bars), taking the protein expression levels and spectral counts of nPcG as 1.
Figure Legend Snippet: Validation of variable protein expression levels based on immunoblotting analyses. Immunoblotting analysis for MRJP2, aldolase, acetyl-CoA acyltransferase 2, and IDGF4 were performed with two independent biologic samples (red and green bars), and the band intensity for each protein in each gland was normalized to that of beta-actin. The normalized protein expression levels in each gland were compared with the spectral counts (SC) of each protein in each gland normalized to that of beta-actin (blue bars), taking the protein expression levels and spectral counts of nPcG as 1.

Techniques Used: Expressing

9) Product Images from "Involvement of CSE/ H2S in high glucose induced aberrant secretion of adipokines in 3T3-L1 adipocytes"

Article Title: Involvement of CSE/ H2S in high glucose induced aberrant secretion of adipokines in 3T3-L1 adipocytes

Journal: Lipids in Health and Disease

doi: 10.1186/1476-511X-13-155

Effect of high glucose on CSE protein expression in 3T3-LI preadipocytes and adipocytes. 3T3-LI preadipocytes and adipocytes were treated in normal glucose (5.5 mmol/L) or high glucose (25mmol/L) for 48h. CSE expression was evaluated by Western blotting. A) Representative result of Western blots (n = 3). B) Quantitative analysis of the results from Western Blots. The value represents the average relative ratio of CSE protein to β-actin in each group (n = 3). * P
Figure Legend Snippet: Effect of high glucose on CSE protein expression in 3T3-LI preadipocytes and adipocytes. 3T3-LI preadipocytes and adipocytes were treated in normal glucose (5.5 mmol/L) or high glucose (25mmol/L) for 48h. CSE expression was evaluated by Western blotting. A) Representative result of Western blots (n = 3). B) Quantitative analysis of the results from Western Blots. The value represents the average relative ratio of CSE protein to β-actin in each group (n = 3). * P

Techniques Used: Expressing, Western Blot

10) Product Images from "The role of cytoplasmic p57 in invasion of hepatocellular carcinoma"

Article Title: The role of cytoplasmic p57 in invasion of hepatocellular carcinoma

Journal: BMC Gastroenterology

doi: 10.1186/s12876-015-0319-x

p57 knockdown in hepatoma cell lines and p57 interaction with LIM domain kinase 1. a Western blot analysis of p57 expression in hepatoma cell lines and a normal liver cell line. b Reverse transcription polymerase chain reaction amplification of p57 mRNA in BEL7402 cells transfected with four p57 shRNAs. c Western blot analysis of p57 expression in BEL7402 cells transfected with four p57 shRNAs. d Reverse transcription polymerase chain reaction amplification of p57 mRNA in BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. e Western blot detection of p57 protein in BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. f Co-immunoprecipitation was used for the detection of the association between p57 and LIM domain kinase 1 in hepatoma cell lysates following different treatments. β-actin was used as an internal control. Glyceraldehyde 3-phosphate dehydrogenase was applied as the internal housekeeping gene control. The expression of each protein was detected by western blot in at least three separate experiments, and representative images are shown. LIMK1, LIM domain kinase 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP, co-immunoprecipitation; WB, western blot
Figure Legend Snippet: p57 knockdown in hepatoma cell lines and p57 interaction with LIM domain kinase 1. a Western blot analysis of p57 expression in hepatoma cell lines and a normal liver cell line. b Reverse transcription polymerase chain reaction amplification of p57 mRNA in BEL7402 cells transfected with four p57 shRNAs. c Western blot analysis of p57 expression in BEL7402 cells transfected with four p57 shRNAs. d Reverse transcription polymerase chain reaction amplification of p57 mRNA in BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. e Western blot detection of p57 protein in BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. f Co-immunoprecipitation was used for the detection of the association between p57 and LIM domain kinase 1 in hepatoma cell lysates following different treatments. β-actin was used as an internal control. Glyceraldehyde 3-phosphate dehydrogenase was applied as the internal housekeeping gene control. The expression of each protein was detected by western blot in at least three separate experiments, and representative images are shown. LIMK1, LIM domain kinase 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP, co-immunoprecipitation; WB, western blot

Techniques Used: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Immunoprecipitation

p57 regulates the expression of p-cofilin in the cytoplasm. a Confocal immunofluorescence microscopy analysis of BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. Green fluorescence represents the transfected plasmid, red fluorescence shows the expression of the target protein, and blue fluorescence shows the nucleus. b Western blot analysis of nuclear and cytoplasmic extracts of p-cofilin in BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. β-actin was used as an internal control for cytoplasmic proteins. Lamin A was used as an internal control for nuclear proteins. GFP, Green fluorescent protein; TRITC, tetramethylrhodamine; DAPI, 4′,6-diamidino-2-phenylindole
Figure Legend Snippet: p57 regulates the expression of p-cofilin in the cytoplasm. a Confocal immunofluorescence microscopy analysis of BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. Green fluorescence represents the transfected plasmid, red fluorescence shows the expression of the target protein, and blue fluorescence shows the nucleus. b Western blot analysis of nuclear and cytoplasmic extracts of p-cofilin in BEL7402-shNC, BEL7402-shp57, SMMC7721-shNC and SMMC7721-shp57 cells. β-actin was used as an internal control for cytoplasmic proteins. Lamin A was used as an internal control for nuclear proteins. GFP, Green fluorescent protein; TRITC, tetramethylrhodamine; DAPI, 4′,6-diamidino-2-phenylindole

Techniques Used: Expressing, Immunofluorescence, Microscopy, Fluorescence, Transfection, Plasmid Preparation, Western Blot

11) Product Images from "miR-620 promotes tumor radioresistance by targeting 15-hydroxyprostaglandin dehydrogenase (HPGD)"

Article Title: miR-620 promotes tumor radioresistance by targeting 15-hydroxyprostaglandin dehydrogenase (HPGD)

Journal: Oncotarget

doi:

HPGD is a target of miR-620, reduces cellular PGE-2 levels and induces radiation resistance A. Representative western blot for HPGD and β-actin (loading control) levels in MDA-MB-231 and DU-145 cells transiently transfected with control (C) or miR-620 (620) mimic. B. MDA-MB-231 and DU145 cells were transiently cotransfected with wildtype (wt) or mutant (mt) HPGD 3′UTR luciferase vector, renilla vector and control or miR-620 mimic, then firefly luciferase assay performed and normalized to renilla signal. Sequences of the miR-620 binding sequence in the HPGD wt and mt 3′UTR are shown. C. MDA-MB-231 and DU145 cells transiently transfected with control or miR-620 mimic, or transiently transfected with control or HPGD siRNA were lysed and subjected to PGE2 assays (representative experiments shown). D. MDA-MB-231 and DU145 cells were transiently transfected with control or HPGD siRNA and radiation clonogenic survival assays performed. E. MDA-MB-231 and DU145 cells were treated with PGE2 or vehicle, irradiated (4 Gy or 6 Gy, respectively) and radiation clonogenic survival assays performed. Mean, standard deviations and statistical significance are denoted; * p
Figure Legend Snippet: HPGD is a target of miR-620, reduces cellular PGE-2 levels and induces radiation resistance A. Representative western blot for HPGD and β-actin (loading control) levels in MDA-MB-231 and DU-145 cells transiently transfected with control (C) or miR-620 (620) mimic. B. MDA-MB-231 and DU145 cells were transiently cotransfected with wildtype (wt) or mutant (mt) HPGD 3′UTR luciferase vector, renilla vector and control or miR-620 mimic, then firefly luciferase assay performed and normalized to renilla signal. Sequences of the miR-620 binding sequence in the HPGD wt and mt 3′UTR are shown. C. MDA-MB-231 and DU145 cells transiently transfected with control or miR-620 mimic, or transiently transfected with control or HPGD siRNA were lysed and subjected to PGE2 assays (representative experiments shown). D. MDA-MB-231 and DU145 cells were transiently transfected with control or HPGD siRNA and radiation clonogenic survival assays performed. E. MDA-MB-231 and DU145 cells were treated with PGE2 or vehicle, irradiated (4 Gy or 6 Gy, respectively) and radiation clonogenic survival assays performed. Mean, standard deviations and statistical significance are denoted; * p

Techniques Used: Western Blot, Multiple Displacement Amplification, Transfection, Mutagenesis, Luciferase, Plasmid Preparation, Binding Assay, Sequencing, Irradiation

12) Product Images from "Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration"

Article Title: Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1511016112

Increased liver damage in TOP1mt KO mice 4 d after exposure to CCl 4 . ( A ) Apoptosis analysis by Western blotting for cleaved caspase-3. β-actin was used as loading control. ( B ) Increased lipid accumulation in TOP1mt KO livers 4 d after CCl 4 administration.
Figure Legend Snippet: Increased liver damage in TOP1mt KO mice 4 d after exposure to CCl 4 . ( A ) Apoptosis analysis by Western blotting for cleaved caspase-3. β-actin was used as loading control. ( B ) Increased lipid accumulation in TOP1mt KO livers 4 d after CCl 4 administration.

Techniques Used: Mouse Assay, Western Blot

13) Product Images from "All-Trans Retinoic Acid Enhances Matrix Metalloproteinase 2 Expression and Secretion in Human Myeloid Leukemia THP-1 Cells"

Article Title: All-Trans Retinoic Acid Enhances Matrix Metalloproteinase 2 Expression and Secretion in Human Myeloid Leukemia THP-1 Cells

Journal: BioMed Research International

doi: 10.1155/2018/5971080

ATRA upregulates MMP-2 mRNA expression but suppresses its cell surface expression of THP-1. THP-1 cells were treated with the different concentrations of ATRA as indicated above for 24 h (a and c) or with 1  μ M (b) or 100 nM (d) ATRA for various time points as indicated above. The expression of MMP-2 mRNA was determined by qRT-PCR and was normalized to that of  β -actin (a and b). The cell surface expression of MMP-2 was analyzed by flow cytometry (c and d). Bar graphs show the relative gene expression ± SD. ∗ P
Figure Legend Snippet: ATRA upregulates MMP-2 mRNA expression but suppresses its cell surface expression of THP-1. THP-1 cells were treated with the different concentrations of ATRA as indicated above for 24 h (a and c) or with 1 μ M (b) or 100 nM (d) ATRA for various time points as indicated above. The expression of MMP-2 mRNA was determined by qRT-PCR and was normalized to that of β -actin (a and b). The cell surface expression of MMP-2 was analyzed by flow cytometry (c and d). Bar graphs show the relative gene expression ± SD. ∗ P

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

14) Product Images from "Enhancement of Autophagy by Simvastatin through Inhibition of Rac1-mTOR Signaling Pathway in Coronary Arterial Myocytes"

Article Title: Enhancement of Autophagy by Simvastatin through Inhibition of Rac1-mTOR Signaling Pathway in Coronary Arterial Myocytes

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

doi: 10.1159/000350111

Simvastatin increase LC3B and calponin expression in coronary arteries. Mice were intragastrically fed simvastatin for two weeks with or without mevalonate. Frozen sections of mouse hearts were stained with Alexa488-anti-α-smooth muscle actin (α-SMA) and Alexa555-anti-LC3B or Alexa555-anti-calponin antibodies. Displayed are representative confocal microscopic images showing the expression of LC3B (A) or calponin (B) in coronary arteries (n=5).
Figure Legend Snippet: Simvastatin increase LC3B and calponin expression in coronary arteries. Mice were intragastrically fed simvastatin for two weeks with or without mevalonate. Frozen sections of mouse hearts were stained with Alexa488-anti-α-smooth muscle actin (α-SMA) and Alexa555-anti-LC3B or Alexa555-anti-calponin antibodies. Displayed are representative confocal microscopic images showing the expression of LC3B (A) or calponin (B) in coronary arteries (n=5).

Techniques Used: Expressing, Mouse Assay, Staining

15) Product Images from "APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *"

Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M708728200

Role of Vif polyubiquitylation in A3G degradation. A , polyubiquitylation of HIV-1 Vif protein in vivo . Wild-type or Lys-free (Vif16K/R) HIV-1 Vif proteins were coexpressed with FLAG-tagged Ub 48A . Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum. B , expression of Lys-free Vif. Equal amounts of expression vectors for wild-type ( WT ) or Lys-free Vif were transfected into 293T cells, and protein expression was determined by Western blotting. C , activity of Lys-free Vif. Wild-type or Lys-free Vif proteins were coexpressed with A3G protein fused with FLuc in 293T cells, and the cellular luciferase activity was determined as before. Error bars represent S.D. in at least three independent experiments. D , interaction of HIV-1 Vif with A3G and Cul5 E3 ligase. Vif proteins were coexpressed with FLAG-tagged A3G or HA-tagged Cul5 or EloC. To detect the interaction between Vif and A3G, proteins were pulled down by anti-FLAG beads and analyzed by Western blotting using anti-FLAG and anti-Vif antibodies. To detect the interaction between Vif and Cul5 or EloC, proteins were immunoprecipitated ( IP ) by anti-Vif polyclonal antibody and analyzed by Western blotting using anti-Vif and anti-HA antibodies. I , input; P , pull down. E , A3G polyubiquitylation by Lys-free Vif. Wild-type A3G was coexpressed with wild-type or Lys-free Vif in the presence of the FLAG-tagged Ub 48A expression vector. Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum.
Figure Legend Snippet: Role of Vif polyubiquitylation in A3G degradation. A , polyubiquitylation of HIV-1 Vif protein in vivo . Wild-type or Lys-free (Vif16K/R) HIV-1 Vif proteins were coexpressed with FLAG-tagged Ub 48A . Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum. B , expression of Lys-free Vif. Equal amounts of expression vectors for wild-type ( WT ) or Lys-free Vif were transfected into 293T cells, and protein expression was determined by Western blotting. C , activity of Lys-free Vif. Wild-type or Lys-free Vif proteins were coexpressed with A3G protein fused with FLuc in 293T cells, and the cellular luciferase activity was determined as before. Error bars represent S.D. in at least three independent experiments. D , interaction of HIV-1 Vif with A3G and Cul5 E3 ligase. Vif proteins were coexpressed with FLAG-tagged A3G or HA-tagged Cul5 or EloC. To detect the interaction between Vif and A3G, proteins were pulled down by anti-FLAG beads and analyzed by Western blotting using anti-FLAG and anti-Vif antibodies. To detect the interaction between Vif and Cul5 or EloC, proteins were immunoprecipitated ( IP ) by anti-Vif polyclonal antibody and analyzed by Western blotting using anti-Vif and anti-HA antibodies. I , input; P , pull down. E , A3G polyubiquitylation by Lys-free Vif. Wild-type A3G was coexpressed with wild-type or Lys-free Vif in the presence of the FLAG-tagged Ub 48A expression vector. Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum.

Techniques Used: In Vivo, Isolation, Western Blot, Expressing, Transfection, Activity Assay, Luciferase, Immunoprecipitation, Plasmid Preparation

16) Product Images from "Critical roles for Nitric oxide and ERK in the completion of prosurvival autophagy in 4OHTAM-treated estrogen receptor-positive breast cancer cells"

Article Title: Critical roles for Nitric oxide and ERK in the completion of prosurvival autophagy in 4OHTAM-treated estrogen receptor-positive breast cancer cells

Journal: Cancer letters

doi: 10.1016/j.canlet.2014.07.031

O2− and NO regulate formation of autophagic vesicles and lysosomal/autolysosomal integrity in MCF7 cells (A) The cells were treated with the 4OHTAM (5 μM) in combination with vehicle or Tiron (2 mM) or c-PTIO (100 μM) or Dea NoNoate (100 μM) for 48 hours. Whole cell lysates were immunoblotted for LC3 with β-actin as loading control. The cells in triplicate were also analyzed for MDC sequestration (B) and AO red/green ratio (C). The average was normalized to the vehicle condition and presented with standard deviation indicated. In B, There is significant difference between 4OHTAM alone and 4OHTAM plus Tiron (p=0.001) or plus c-PTIO (p
Figure Legend Snippet: O2− and NO regulate formation of autophagic vesicles and lysosomal/autolysosomal integrity in MCF7 cells (A) The cells were treated with the 4OHTAM (5 μM) in combination with vehicle or Tiron (2 mM) or c-PTIO (100 μM) or Dea NoNoate (100 μM) for 48 hours. Whole cell lysates were immunoblotted for LC3 with β-actin as loading control. The cells in triplicate were also analyzed for MDC sequestration (B) and AO red/green ratio (C). The average was normalized to the vehicle condition and presented with standard deviation indicated. In B, There is significant difference between 4OHTAM alone and 4OHTAM plus Tiron (p=0.001) or plus c-PTIO (p

Techniques Used: Standard Deviation

C-PTIO inhibits chloroquine-induced formation of autophagic vesicles without affecting LC3 expression in MCF7 cells The cells were treated with vehicle or 4OHTAM (5 μM) in combination with c-PTIO (5 μM), chloroquine (10 μM), or c-PTIO/chloroquine. Whole cell lysates were immunoblotted for LC-3 and β-actin (A). The cells were analyzed for MDC sequestration (B). There is significant difference (p
Figure Legend Snippet: C-PTIO inhibits chloroquine-induced formation of autophagic vesicles without affecting LC3 expression in MCF7 cells The cells were treated with vehicle or 4OHTAM (5 μM) in combination with c-PTIO (5 μM), chloroquine (10 μM), or c-PTIO/chloroquine. Whole cell lysates were immunoblotted for LC-3 and β-actin (A). The cells were analyzed for MDC sequestration (B). There is significant difference (p

Techniques Used: Expressing

17) Product Images from "The effects of buthionine sulfoximine on the proliferation and apoptosis of biliary tract cancer cells induced by cisplatin and gemcitabine"

Article Title: The effects of buthionine sulfoximine on the proliferation and apoptosis of biliary tract cancer cells induced by cisplatin and gemcitabine

Journal: Oncology Letters

doi: 10.3892/ol.2015.3879

Effect of BSO on antiapoptotic protein expression in BTC cells immunoblot analysis of Mcl-1, Bcl-xL and Bcl-2 expression in (A) GBC-SD and (B) RBE cells. Cells were treated with 50 µM BSO and harvested at the indicated times. β-actin was
Figure Legend Snippet: Effect of BSO on antiapoptotic protein expression in BTC cells immunoblot analysis of Mcl-1, Bcl-xL and Bcl-2 expression in (A) GBC-SD and (B) RBE cells. Cells were treated with 50 µM BSO and harvested at the indicated times. β-actin was

Techniques Used: Expressing

18) Product Images from "Prolylcarboxypeptidase Regulates Proliferation, Autophagy, and Resistance to 4-Hydroxytamoxifen-induced Cytotoxicity in Estrogen Receptor-positive Breast Cancer Cells *"

Article Title: Prolylcarboxypeptidase Regulates Proliferation, Autophagy, and Resistance to 4-Hydroxytamoxifen-induced Cytotoxicity in Estrogen Receptor-positive Breast Cancer Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.143271

Autophagy is inhibited by ZPP in MCF7s, B6-9, and the TRC cells, which show higher autophagy activity. A and B , MCF7 ( A ) and B6-9 ( B ) cells were treated with 4OHTAM/vehicle and 4OHTAM/ZPP for 2 days. Whole cell lysates were immunoblotted for LC3 and β-actin (representative picture of three independent experiments). C , MCF7 cell and the TRC cells were treated with 4OHTAM for 2 days. Whole cell lysates were immunoblotted for LC3 and β-actin (representative picture of three independent experiments). D , TRC cells were treated with 4OHTAM/vehicle and 4OHTAM/ZPP for 2 days. Whole cell lysates were immunoblotted for LC3 and β-actin (representative picture of three independent experiments). E , MCF7 and TRC cells were treated with 4OHTAM/vehicle and 4OHTAM/ZPP for 2 days. MDC sequestration assay was performed. The average MDC fluorescence in triplicate was normalized to the vehicle treated MCF7 cells and presented as a graph with standard deviation as error bars (representative graph of three independent experiments).
Figure Legend Snippet: Autophagy is inhibited by ZPP in MCF7s, B6-9, and the TRC cells, which show higher autophagy activity. A and B , MCF7 ( A ) and B6-9 ( B ) cells were treated with 4OHTAM/vehicle and 4OHTAM/ZPP for 2 days. Whole cell lysates were immunoblotted for LC3 and β-actin (representative picture of three independent experiments). C , MCF7 cell and the TRC cells were treated with 4OHTAM for 2 days. Whole cell lysates were immunoblotted for LC3 and β-actin (representative picture of three independent experiments). D , TRC cells were treated with 4OHTAM/vehicle and 4OHTAM/ZPP for 2 days. Whole cell lysates were immunoblotted for LC3 and β-actin (representative picture of three independent experiments). E , MCF7 and TRC cells were treated with 4OHTAM/vehicle and 4OHTAM/ZPP for 2 days. MDC sequestration assay was performed. The average MDC fluorescence in triplicate was normalized to the vehicle treated MCF7 cells and presented as a graph with standard deviation as error bars (representative graph of three independent experiments).

Techniques Used: Activity Assay, Fluorescence, Standard Deviation

19) Product Images from "Guanosine effect on cholesterol efflux and apolipoprotein E expression in astrocytes"

Article Title: Guanosine effect on cholesterol efflux and apolipoprotein E expression in astrocytes

Journal: Purinergic Signalling

doi: 10.1007/s11302-006-9011-5

Time course of ABCA1 expression induced by 150 µM guanosine in (a) C6 rat glioma cells and (b) rat brain cultured astrocytes. Cells were serum deprived for 24 h and were treated for the indicated periods with 150 µM guanosine or with ApoA1 (15 µg/ml) plus 22R (10 µg/ml) and RA (10 µg/ml). Fifty microgrammes of total protein were loaded per lane and immunoblotted with rabbit polyclonal antibody to ABCA1. Western blotting with β actin antibody was used as a loading control. The blots are representative of three independent experiments with similar results. Immunoblots were quantified by densitometric analysis, and the ABCA1 values, normalised to the corresponding β actin values, are expressed as number of times of increase versus basal values (untreated cells) in the histograms under the blots. Data are mean ± SEM of three independent experiments. * P
Figure Legend Snippet: Time course of ABCA1 expression induced by 150 µM guanosine in (a) C6 rat glioma cells and (b) rat brain cultured astrocytes. Cells were serum deprived for 24 h and were treated for the indicated periods with 150 µM guanosine or with ApoA1 (15 µg/ml) plus 22R (10 µg/ml) and RA (10 µg/ml). Fifty microgrammes of total protein were loaded per lane and immunoblotted with rabbit polyclonal antibody to ABCA1. Western blotting with β actin antibody was used as a loading control. The blots are representative of three independent experiments with similar results. Immunoblots were quantified by densitometric analysis, and the ABCA1 values, normalised to the corresponding β actin values, are expressed as number of times of increase versus basal values (untreated cells) in the histograms under the blots. Data are mean ± SEM of three independent experiments. * P

Techniques Used: Expressing, Cell Culture, Western Blot

Expression of ABCA1 in rat brain cultured astrocytes and C6 rat glioma cells was investigated by RT-PCR and by Western blot. (a) Total RNA was isolated from the cells, and 2.5 µg was used for reverse transcription and then subjected to PCR amplification using primer pairs specific for this transporter. Amplification products of the expected size (331 bp) were resolved by agarose (1.5%) gel electrophoresis. M represents the size markers as indicated; lane 1 astrocytes, lane 2 C6 cells. (b) Total proteins were isolated from the cells, and equal amounts (50 µg) were separated on 12% SDS-polyacrylamide gel. ABCA1 protein was detected using a rabbit polyclonal anti-ABCA1 antibody. The immunoblot was also stripped, and western blotting with β actin antibody was used as a loading control. Lane 1 astrocytes; lane 2 C6 cells. Results are representative of experiments carried out with RNA and protein isolated from at least three independent cell culture seedings
Figure Legend Snippet: Expression of ABCA1 in rat brain cultured astrocytes and C6 rat glioma cells was investigated by RT-PCR and by Western blot. (a) Total RNA was isolated from the cells, and 2.5 µg was used for reverse transcription and then subjected to PCR amplification using primer pairs specific for this transporter. Amplification products of the expected size (331 bp) were resolved by agarose (1.5%) gel electrophoresis. M represents the size markers as indicated; lane 1 astrocytes, lane 2 C6 cells. (b) Total proteins were isolated from the cells, and equal amounts (50 µg) were separated on 12% SDS-polyacrylamide gel. ABCA1 protein was detected using a rabbit polyclonal anti-ABCA1 antibody. The immunoblot was also stripped, and western blotting with β actin antibody was used as a loading control. Lane 1 astrocytes; lane 2 C6 cells. Results are representative of experiments carried out with RNA and protein isolated from at least three independent cell culture seedings

Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

20) Product Images from "Activation and expression of μ-calpain in dorsal root contributes to RTX-induced mechanical allodynia"

Article Title: Activation and expression of μ-calpain in dorsal root contributes to RTX-induced mechanical allodynia

Journal: Molecular Pain

doi: 10.1177/1744806917719169

The effect of calpain inhibitor MDL 28170 on RTX induced calpain activation and degradation of myelin basic protein (MBP) in L4–L6 dorsal root. (a) to (c) Representative gel images show the protein level of µ-calpain, α II-spectrin, α II-spectrin breakdown product (spectrin BD), and 21.5- and 18.5-kDa MBP isoform in L4–L6 dorsal root obtained from vehicle, RTX 6 W, MDL 28170 (MDL), and PEG400/DMSO (P/D) rats. β-actin was used as a loading control. (d), (g), (f) Summary data show the protein level of µ-calpain and 21.5- and 18.5-kDa MBP isoform in L4–L6 dorsal root. (e) Summary data show the percentage of α II-spectrin breakdown product in the total of α II-spectrin in L4–L6 dorsal root. Data are expressed as means ± SEM (n = 6 rats in each group). *P
Figure Legend Snippet: The effect of calpain inhibitor MDL 28170 on RTX induced calpain activation and degradation of myelin basic protein (MBP) in L4–L6 dorsal root. (a) to (c) Representative gel images show the protein level of µ-calpain, α II-spectrin, α II-spectrin breakdown product (spectrin BD), and 21.5- and 18.5-kDa MBP isoform in L4–L6 dorsal root obtained from vehicle, RTX 6 W, MDL 28170 (MDL), and PEG400/DMSO (P/D) rats. β-actin was used as a loading control. (d), (g), (f) Summary data show the protein level of µ-calpain and 21.5- and 18.5-kDa MBP isoform in L4–L6 dorsal root. (e) Summary data show the percentage of α II-spectrin breakdown product in the total of α II-spectrin in L4–L6 dorsal root. Data are expressed as means ± SEM (n = 6 rats in each group). *P

Techniques Used: Activation Assay

Identification of myelin basic protein (MBP) isoforms degradation in L4–L6 dorsal root. (a) Representative gel images show the total protein level of 21.5- and 18.5-kDa MBP isoforms in L4–L6 dorsal root obtained from vehicle group and RTX group rats. β-actin was used as a loading control. (b) and (c) Summary data show the total protein level of 21.5- and 18.5-kDa MBP isoforms in L4–L6 dorsal root. Data are expressed as means ± SEM (n = 8 rats in each group). *P
Figure Legend Snippet: Identification of myelin basic protein (MBP) isoforms degradation in L4–L6 dorsal root. (a) Representative gel images show the total protein level of 21.5- and 18.5-kDa MBP isoforms in L4–L6 dorsal root obtained from vehicle group and RTX group rats. β-actin was used as a loading control. (b) and (c) Summary data show the total protein level of 21.5- and 18.5-kDa MBP isoforms in L4–L6 dorsal root. Data are expressed as means ± SEM (n = 8 rats in each group). *P

Techniques Used:

Quantitative analysis of the expression and activation of µ-calpain in L4–L6 dorsal root. (a) and (b) Representative gel images show the protein level of µ-calpain, α II-spectrin, and α II-spectrin breakdown product (spectrin BD) in L4–L6 dorsal root obtained from vehicle group and RTX group rats. β-actin was used as a loading control. (c) Summary data show the protein level of µ-calpain in L4–L6 dorsal root. (d) Summary data show the percentage of α II-spectrin breakdown product in the total of α II-spectrin in L4–L6 dorsal root. The calpain activity was estimated by evaluating the protein level of α II-spectrin breakdown product (150 kDa) using Western blotting. Data are expressed as means ± SEM (n = 8 rats in each group). *P
Figure Legend Snippet: Quantitative analysis of the expression and activation of µ-calpain in L4–L6 dorsal root. (a) and (b) Representative gel images show the protein level of µ-calpain, α II-spectrin, and α II-spectrin breakdown product (spectrin BD) in L4–L6 dorsal root obtained from vehicle group and RTX group rats. β-actin was used as a loading control. (c) Summary data show the protein level of µ-calpain in L4–L6 dorsal root. (d) Summary data show the percentage of α II-spectrin breakdown product in the total of α II-spectrin in L4–L6 dorsal root. The calpain activity was estimated by evaluating the protein level of α II-spectrin breakdown product (150 kDa) using Western blotting. Data are expressed as means ± SEM (n = 8 rats in each group). *P

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

21) Product Images from "Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish"

Article Title: Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-015-0207-2

Constitutively activated MEK1 (MEK1 S219D ) and MEK2 (MEK2 S219D ) both phosphorylated ERK1. a . COS-1 cells were transiently transfected with pcDNA3-HA, pcDNA3-ERK1-HA, pcDNA3-MEK1-mCherry, pcDNA3-MEK1S219D-GFP, pcDNA3-MEK2-GFP, or pcDNA3-MEK2S219D-GFP. Total lysates were analyzed by Western blotting using anti-HA, anti-GFP, anti-pERK monoclonal antibodies; anti-mCherry and anti-Actin polyclonal antibodies. b . Intracellular localization of ERK1, MEK1, and MEK2 in COS-1 cells by fluorescent microscopy. The pcDNA3-ERK1-HA was co-transfected with pcDNA3-GFP (A1-A4), pcDNA3-MEK1-mCherry (B1-B4), pcDNA3-MEK1S219D-GFP (C1-C4), pcDNA3-MEK2-GFP (D1-D4), or pcDNA3-MEK2S219D-GFP (E1-E4). Cy2 or Cy3 dye used an anti-HA monoclonal antibody to detect localization of ERK1 as visualized. DAPI was used to stain nuclear DNA. White arrows indicate the ERK1 protein localized in nuclei and the cytoplasm. IB, immunoblot
Figure Legend Snippet: Constitutively activated MEK1 (MEK1 S219D ) and MEK2 (MEK2 S219D ) both phosphorylated ERK1. a . COS-1 cells were transiently transfected with pcDNA3-HA, pcDNA3-ERK1-HA, pcDNA3-MEK1-mCherry, pcDNA3-MEK1S219D-GFP, pcDNA3-MEK2-GFP, or pcDNA3-MEK2S219D-GFP. Total lysates were analyzed by Western blotting using anti-HA, anti-GFP, anti-pERK monoclonal antibodies; anti-mCherry and anti-Actin polyclonal antibodies. b . Intracellular localization of ERK1, MEK1, and MEK2 in COS-1 cells by fluorescent microscopy. The pcDNA3-ERK1-HA was co-transfected with pcDNA3-GFP (A1-A4), pcDNA3-MEK1-mCherry (B1-B4), pcDNA3-MEK1S219D-GFP (C1-C4), pcDNA3-MEK2-GFP (D1-D4), or pcDNA3-MEK2S219D-GFP (E1-E4). Cy2 or Cy3 dye used an anti-HA monoclonal antibody to detect localization of ERK1 as visualized. DAPI was used to stain nuclear DNA. White arrows indicate the ERK1 protein localized in nuclei and the cytoplasm. IB, immunoblot

Techniques Used: Transfection, Western Blot, Microscopy, Staining

22) Product Images from "Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *"

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.175968

Nedd4-2 decreased TRPV6 stability. X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA alone or together with 12.5 ng of Nedd4-2 cRNA. Two days after injection, the stability of TRPV6 protein was examined by treating the oocytes with 100 μg/ml cycloheximide, an inhibitor of protein synthesis, for 0, 3, 6, or 9 h. A , representative Western blot analyses show the level of TRPV6 proteins at each time point in the presence and absence of Nedd4-2. The level of β-actin determined by Western blotting was used as a control for equal loading. B , assessment of the linear range of TRPV6 band intensity. Oocyte lysates containing TRPV6 protein were loaded at varying levels in different lanes and were run together with samples in A , and the linear range of TRPV6 protein level and band intensity were determined. Shown are a representative Western blot for TRPV6 ( top ) and the derived relationship of band intensity and TRPV6 protein level ( bottom ). TRPV6 band intensity is expressed as a percentage of that at 100% input. C , rate of TRPV6 protein degradation in the presence or absence of Nedd4-2. Protein level is expressed as a percentage of band intensity at time 0. Data from seven blots are shown as means ± S.E. ( error bars ). *, p
Figure Legend Snippet: Nedd4-2 decreased TRPV6 stability. X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA alone or together with 12.5 ng of Nedd4-2 cRNA. Two days after injection, the stability of TRPV6 protein was examined by treating the oocytes with 100 μg/ml cycloheximide, an inhibitor of protein synthesis, for 0, 3, 6, or 9 h. A , representative Western blot analyses show the level of TRPV6 proteins at each time point in the presence and absence of Nedd4-2. The level of β-actin determined by Western blotting was used as a control for equal loading. B , assessment of the linear range of TRPV6 band intensity. Oocyte lysates containing TRPV6 protein were loaded at varying levels in different lanes and were run together with samples in A , and the linear range of TRPV6 protein level and band intensity were determined. Shown are a representative Western blot for TRPV6 ( top ) and the derived relationship of band intensity and TRPV6 protein level ( bottom ). TRPV6 band intensity is expressed as a percentage of that at 100% input. C , rate of TRPV6 protein degradation in the presence or absence of Nedd4-2. Protein level is expressed as a percentage of band intensity at time 0. Data from seven blots are shown as means ± S.E. ( error bars ). *, p

Techniques Used: Injection, Western Blot, Derivative Assay

Nedd4-2 dose-dependently decreased TRPV6 protein abundance ( A ) and TRPV6-mediated Ca 2+ uptake ( B ). Groups of X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA with 0, 3.1, 6.3, 12.5, or 25 ng of Nedd4-2 cRNA, and Ca 2+ uptake experiments were performed 2 days later. Data are presented as a percentage of Ca 2+ uptake of the group injected with TRPV6 cRNA alone. Representative Western blot analyses show that TRPV6 protein level decreased as Nedd4-2 protein level increased, and β-actin (loading control) was at a similar level in all of the groups ( A ). Error bars , S.E.
Figure Legend Snippet: Nedd4-2 dose-dependently decreased TRPV6 protein abundance ( A ) and TRPV6-mediated Ca 2+ uptake ( B ). Groups of X. laevis oocytes were injected with 12.5 ng of TRPV6 cRNA with 0, 3.1, 6.3, 12.5, or 25 ng of Nedd4-2 cRNA, and Ca 2+ uptake experiments were performed 2 days later. Data are presented as a percentage of Ca 2+ uptake of the group injected with TRPV6 cRNA alone. Representative Western blot analyses show that TRPV6 protein level decreased as Nedd4-2 protein level increased, and β-actin (loading control) was at a similar level in all of the groups ( A ). Error bars , S.E.

Techniques Used: Injection, Western Blot

23) Product Images from "GPC1 exosome and its regulatory mi RNAs are specific markers for the detection and target therapy of colorectal cancer"

Article Title: GPC1 exosome and its regulatory mi RNAs are specific markers for the detection and target therapy of colorectal cancer

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12941

Isolation of GPC 1 + exosomes. ( A ) Representative Western blots of GPC 1 and β‐actin protein expression in tissues. ( B ) Semi‐quantitative analysis of GPC 1 protein expression in human normal colon tissues ( n = 89) and CRC tumour tissues ( n = 102). ** P
Figure Legend Snippet: Isolation of GPC 1 + exosomes. ( A ) Representative Western blots of GPC 1 and β‐actin protein expression in tissues. ( B ) Semi‐quantitative analysis of GPC 1 protein expression in human normal colon tissues ( n = 89) and CRC tumour tissues ( n = 102). ** P

Techniques Used: Isolation, Gel Permeation Chromatography, Western Blot, Expressing

24) Product Images from "Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells"

Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells

Journal: Journal of Thoracic Disease

doi: 10.21037/jtd.2019.07.55

Effect of miR-181a-2-3p on inflammasome activation. miR-181a-2-3p promotes inflammasome activation in human THP-1-derived macrophages. Human THP-1-derived macrophages were pretreated with miR-181a-2-3p mimic/inhibitor (50 nM) and then stimulated with 2 mM ATP, 500 µg/mL flagellin, or 2 µg/mL dsDNA to activate NLRP3, NLRC4 and AIM2 inflammasomes, respectively. (A,B) Western blot analysis of proteins associated with the activation of NLRP3, NLRC4 and AIM2 inflammasomes, such as IL-1β and caspase 1. (C) Quantitation of secreted IL-1β band intensities and their normalization with internal control β-actin by Image J software. All data shown are representative of at least three independent experiments. *, P
Figure Legend Snippet: Effect of miR-181a-2-3p on inflammasome activation. miR-181a-2-3p promotes inflammasome activation in human THP-1-derived macrophages. Human THP-1-derived macrophages were pretreated with miR-181a-2-3p mimic/inhibitor (50 nM) and then stimulated with 2 mM ATP, 500 µg/mL flagellin, or 2 µg/mL dsDNA to activate NLRP3, NLRC4 and AIM2 inflammasomes, respectively. (A,B) Western blot analysis of proteins associated with the activation of NLRP3, NLRC4 and AIM2 inflammasomes, such as IL-1β and caspase 1. (C) Quantitation of secreted IL-1β band intensities and their normalization with internal control β-actin by Image J software. All data shown are representative of at least three independent experiments. *, P

Techniques Used: Activation Assay, Derivative Assay, Western Blot, Quantitation Assay, Software

25) Product Images from "Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition"

Article Title: Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.227

miR-185 negatively regulates ATR expression at post-transcriptional level. ( a ) Construction of a vector with either the wild-type sequence of the 3′-UTR of ATR mRNA (ATR-3′-UTR) or a mutated seed sequence of the miR-185-binding site (ATR-3′-UTR-mut). The seed sequence is shown in red. ( b ) Luciferase reporter assays. Each constructed vector was co-transfected with exogenous pre-miR-185 or pre-neg into 786-O cells. Luciferase activity was read 24 h after transfection. ( c ) ATR expression regulated by miR-185 at the mRNA level. qRT-PCR was conducted to quantify the expression level of ATR mRNA at the indicated time points after 786-O cells were transfected with pre-miR-185 or pre-neg. ( d ) ATR expression regulated by miR-185 at the protein level. Western blotting was performed at the indicated time points after transfection with pre-miR-185 or pre-neg.  β -Actin, loading control. ( e ) Densitometric analysis of ATR protein levels shown in panel  d . Each experiment was conducted at least three times independently. * P
Figure Legend Snippet: miR-185 negatively regulates ATR expression at post-transcriptional level. ( a ) Construction of a vector with either the wild-type sequence of the 3′-UTR of ATR mRNA (ATR-3′-UTR) or a mutated seed sequence of the miR-185-binding site (ATR-3′-UTR-mut). The seed sequence is shown in red. ( b ) Luciferase reporter assays. Each constructed vector was co-transfected with exogenous pre-miR-185 or pre-neg into 786-O cells. Luciferase activity was read 24 h after transfection. ( c ) ATR expression regulated by miR-185 at the mRNA level. qRT-PCR was conducted to quantify the expression level of ATR mRNA at the indicated time points after 786-O cells were transfected with pre-miR-185 or pre-neg. ( d ) ATR expression regulated by miR-185 at the protein level. Western blotting was performed at the indicated time points after transfection with pre-miR-185 or pre-neg. β -Actin, loading control. ( e ) Densitometric analysis of ATR protein levels shown in panel d . Each experiment was conducted at least three times independently. * P

Techniques Used: Expressing, Plasmid Preparation, Sequencing, Binding Assay, Luciferase, Construct, Transfection, Activity Assay, Quantitative RT-PCR, Western Blot

miR-185 enhances apoptosis and proliferation inhibition by negatively regulating ATR. ( a ) ATR, ATM, Chk1 and pChk1 expression at the protein level. Western blotting was performed to measure the protein levels of ATR, ATM, Chk1 and pChk1 in cells transfected with pre-miR-185 (P185), pre-neg (PN) or without transfection (Ctrl) 24 h after exposure of 786-O cells to 0 and 4 Gy X-rays.  β -Actin, loading control. ( b ) ATR and ATM expression at the mRNA level. qRT-PCR was conducted to quantify mRNA of ATR and ATM in cells transfected with pre-miR-185 or pre-neg 24 h after exposure of 786-O cells to 4 Gy X-rays. ( c ) Apoptotic rate of 786-O cells analyzed by flow cytometry 24 h after X-ray or carbon ion irradiation. ( d ) DNA histograms where the relative cell counts were plotted against DNA content 24 h after cells were exposed to UVB and stained with propidium iodide. Sub-G1 (gate M1) demonstrates the apoptotic cells. One of three independent experiments is shown. ( e ) Micrographs of EdU-positive 786-O cells 24 h after exposure to 2 Gy of X-rays. Scale bar, 25  μ m. ( f ) Bar charts for the fraction of EdU-positive cells after various treatments. ( g ) Micrographs of the density and morphology of 786-O cells 48 h after exposure to 4 Gy of X-rays; Scale bar, 100  μ m. ( h ) Growth curves of 786-O cells after various treatments. Each experiment was conducted at least three times independently except that carbon ion irradiation was conducted two times because of the beam time restriction and did not show error bars in the histograms. * P
Figure Legend Snippet: miR-185 enhances apoptosis and proliferation inhibition by negatively regulating ATR. ( a ) ATR, ATM, Chk1 and pChk1 expression at the protein level. Western blotting was performed to measure the protein levels of ATR, ATM, Chk1 and pChk1 in cells transfected with pre-miR-185 (P185), pre-neg (PN) or without transfection (Ctrl) 24 h after exposure of 786-O cells to 0 and 4 Gy X-rays. β -Actin, loading control. ( b ) ATR and ATM expression at the mRNA level. qRT-PCR was conducted to quantify mRNA of ATR and ATM in cells transfected with pre-miR-185 or pre-neg 24 h after exposure of 786-O cells to 4 Gy X-rays. ( c ) Apoptotic rate of 786-O cells analyzed by flow cytometry 24 h after X-ray or carbon ion irradiation. ( d ) DNA histograms where the relative cell counts were plotted against DNA content 24 h after cells were exposed to UVB and stained with propidium iodide. Sub-G1 (gate M1) demonstrates the apoptotic cells. One of three independent experiments is shown. ( e ) Micrographs of EdU-positive 786-O cells 24 h after exposure to 2 Gy of X-rays. Scale bar, 25  μ m. ( f ) Bar charts for the fraction of EdU-positive cells after various treatments. ( g ) Micrographs of the density and morphology of 786-O cells 48 h after exposure to 4 Gy of X-rays; Scale bar, 100  μ m. ( h ) Growth curves of 786-O cells after various treatments. Each experiment was conducted at least three times independently except that carbon ion irradiation was conducted two times because of the beam time restriction and did not show error bars in the histograms. * P

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Cytometry, Irradiation, Staining

26) Product Images from "The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞"

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M808879200

Expression of JAK2 L611S mutant significantly inhibits apoptosis induced by cytokine removal. A , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml) for the indicated times. The viability of these cells was determined by the trypan blue exclusion method. Results represent the mean ± S.D. of three independent experiments. B , cells were fixed, treated with propidium iodide, and subjected to fluorescence-activated cell sorting analysis as described under “Experimental Procedures.” C , caspase activities were measured by the cleavage of substrates, Ac-DEVD-7-amino-4-methycoumarin for caspase-3 or Ac-LEHD-7-amino-4-methycoumarin for caspase-9, respectively. Results represent the mean ± S.D. of three independent experiments. D , DNA was isolated from cells and subjected to agarose gel electrophoresis. E , XIAP, cIAP, and Bcl-XL mRNAs were determined by reverse transcription-PCR analysis. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA was used as an internal control. F , whole cell lysates were immunoblotted ( IB ) with anti-XIAP antibody, anti-cIAP1 antibody, anti-Bcl-XL antibody, or anti-β-actin antibody.
Figure Legend Snippet: Expression of JAK2 L611S mutant significantly inhibits apoptosis induced by cytokine removal. A , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml) for the indicated times. The viability of these cells was determined by the trypan blue exclusion method. Results represent the mean ± S.D. of three independent experiments. B , cells were fixed, treated with propidium iodide, and subjected to fluorescence-activated cell sorting analysis as described under “Experimental Procedures.” C , caspase activities were measured by the cleavage of substrates, Ac-DEVD-7-amino-4-methycoumarin for caspase-3 or Ac-LEHD-7-amino-4-methycoumarin for caspase-9, respectively. Results represent the mean ± S.D. of three independent experiments. D , DNA was isolated from cells and subjected to agarose gel electrophoresis. E , XIAP, cIAP, and Bcl-XL mRNAs were determined by reverse transcription-PCR analysis. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA was used as an internal control. F , whole cell lysates were immunoblotted ( IB ) with anti-XIAP antibody, anti-cIAP1 antibody, anti-Bcl-XL antibody, or anti-β-actin antibody.

Techniques Used: Expressing, Mutagenesis, Fluorescence, FACS, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction

ALL-derived L611S mutation induces constitutive activation of JAK2. A , scheme of JAK2 L611S mutant ( upper ). BaF3 cell lines were infected with retrovirus encoding WT c-HA or JAK2 mutant c-HA (L611S) and erythropoietin receptor c-FLAG. Cell lysates were immunoblotted ( IB ) with anti-HA antibody, anti-JAK2 antibody, anti-FLAG antibody, or anti-β-actin antibody. MSCV indicates empty virus. B and C , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml) for 24 h. B , cell lysates were subjected to immunoprecipitation ( IP ) using an anti-HA antibody and immunoblotted with antibodies to anti-phospho ( P )-Tyr 1007/1008 JAK2 or anti-HA antibody. C , cell lysates were immunoblotted with anti-phospho-Thr 202 /Tyr 204 ERK antibody, anti-ERK antibody, anti-phospho-Ser 473 Akt antibody, anti-Akt antibody, anti-phospho-Tyr 694 Stat5 antibody, or anti-Stat5 antibody. D , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml). Surviving cells were counted using a Beckman Coulter Vi-CELL at the indicated times. Results represent the mean ± S.D. of three independent experiments. E , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml) for 24 h. Cell proliferation was measured by determining the BrdUrd ( BrdU ) incorporation. Results represent the mean ± S.D. of three independent experiments.
Figure Legend Snippet: ALL-derived L611S mutation induces constitutive activation of JAK2. A , scheme of JAK2 L611S mutant ( upper ). BaF3 cell lines were infected with retrovirus encoding WT c-HA or JAK2 mutant c-HA (L611S) and erythropoietin receptor c-FLAG. Cell lysates were immunoblotted ( IB ) with anti-HA antibody, anti-JAK2 antibody, anti-FLAG antibody, or anti-β-actin antibody. MSCV indicates empty virus. B and C , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml) for 24 h. B , cell lysates were subjected to immunoprecipitation ( IP ) using an anti-HA antibody and immunoblotted with antibodies to anti-phospho ( P )-Tyr 1007/1008 JAK2 or anti-HA antibody. C , cell lysates were immunoblotted with anti-phospho-Thr 202 /Tyr 204 ERK antibody, anti-ERK antibody, anti-phospho-Ser 473 Akt antibody, anti-Akt antibody, anti-phospho-Tyr 694 Stat5 antibody, or anti-Stat5 antibody. D , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml). Surviving cells were counted using a Beckman Coulter Vi-CELL at the indicated times. Results represent the mean ± S.D. of three independent experiments. E , transduced BaF3 cells were washed twice with PBS and left untreated or were stimulated with Epo (5 units/ml) for 24 h. Cell proliferation was measured by determining the BrdUrd ( BrdU ) incorporation. Results represent the mean ± S.D. of three independent experiments.

Techniques Used: Derivative Assay, Mutagenesis, Activation Assay, Infection, Immunoprecipitation, BrdU Incorporation Assay

27) Product Images from "The N-Ethyl-N-Nitrosourea-Induced Goldenticket Mouse Mutant Reveals an Essential Function of Sting in the In Vivo Interferon Response to Listeria monocytogenes and Cyclic Dinucleotides ▿"

Article Title: The N-Ethyl-N-Nitrosourea-Induced Goldenticket Mouse Mutant Reveals an Essential Function of Sting in the In Vivo Interferon Response to Listeria monocytogenes and Cyclic Dinucleotides ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.00999-10

T596A mutation abrogates the function of Sting . (A) Luciferase production was measured 24 h after cotransfection of HEK293T cells with an IFN-β luciferase reporter and increasing concentrations (50 ng, 100 ng, 200 ng per well) of wild-type or Gt Sting . pGL3 is a positive-control luciferase expression vector. (B) Following infection with wild-type L. monocytogenes (L.m.; MOI, 1), infection with Sendai virus (SeV; 150 HAU/ml), or transfection of 500 μg/ml of poly(dAT:dTA) or poly(I:C) for 4 h, RNA was harvested from bone marrow-derived macrophages and the amounts of IFN-β transcripts were measured relative to those of β-actin transcripts. (C) Whole-cell lysates were collected and analyzed for Sting expression by Western blotting following 4 h of treatment with either 4 μg/ml of c-di-GMP or 100 ng/ml of LPS. (D) Whole-cell lysates were collected from HEK293T cells 24 h after transfection with 200 ng/ml of wild-type or Gt Sting . Sting was detected using anti-HA antibody, and β-actin was used as a loading control. (E and F) Sting +/+ or Sting Gt / Gt immortalized bone marrow-derived macrophages were transduced with either empty MSCV2.2 vector, WT Sting , or Gt Sting . (E) Transduced cells were transfected with 500 μg/ml poly(dAT:dTA) or 500 μg/ml purified bacterial ( Legionella pneumophila ) genomic DNA for 4 h, and then RNA was harvested and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. (F) Transduced cells were transfected with 500 μg/ml poly(I:C), infected with Sendai virus at 150 HAU/ml, or treated with 100 ng/ml of LPS for 4 h; RNA was harvested; and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. Data are representative of those from at least three independent experiments. *, P
Figure Legend Snippet: T596A mutation abrogates the function of Sting . (A) Luciferase production was measured 24 h after cotransfection of HEK293T cells with an IFN-β luciferase reporter and increasing concentrations (50 ng, 100 ng, 200 ng per well) of wild-type or Gt Sting . pGL3 is a positive-control luciferase expression vector. (B) Following infection with wild-type L. monocytogenes (L.m.; MOI, 1), infection with Sendai virus (SeV; 150 HAU/ml), or transfection of 500 μg/ml of poly(dAT:dTA) or poly(I:C) for 4 h, RNA was harvested from bone marrow-derived macrophages and the amounts of IFN-β transcripts were measured relative to those of β-actin transcripts. (C) Whole-cell lysates were collected and analyzed for Sting expression by Western blotting following 4 h of treatment with either 4 μg/ml of c-di-GMP or 100 ng/ml of LPS. (D) Whole-cell lysates were collected from HEK293T cells 24 h after transfection with 200 ng/ml of wild-type or Gt Sting . Sting was detected using anti-HA antibody, and β-actin was used as a loading control. (E and F) Sting +/+ or Sting Gt / Gt immortalized bone marrow-derived macrophages were transduced with either empty MSCV2.2 vector, WT Sting , or Gt Sting . (E) Transduced cells were transfected with 500 μg/ml poly(dAT:dTA) or 500 μg/ml purified bacterial ( Legionella pneumophila ) genomic DNA for 4 h, and then RNA was harvested and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. (F) Transduced cells were transfected with 500 μg/ml poly(I:C), infected with Sendai virus at 150 HAU/ml, or treated with 100 ng/ml of LPS for 4 h; RNA was harvested; and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. Data are representative of those from at least three independent experiments. *, P

Techniques Used: Mutagenesis, Luciferase, Cotransfection, Positive Control, Expressing, Plasmid Preparation, Infection, Transfection, Derivative Assay, Western Blot, Transduction, Purification

Sting is required for cyclic dinucleotide-induced IFN-β production. Sting +/+ , Sting Gt / Gt , or Sting − / − bone marrow-derived macrophages were treated with 4 μg/ml c-di-GMP (A) or c-di-AMP (B) for 4 h. RNA was harvested, and the amounts of IFN-β transcripts were measured relative to those of rps17 (A) or β-actin (B) transcripts. (C) Sting +/+ or Sting Gt / Gt immortalized bone marrow-derived macrophages were transduced with either empty MSCV2.2 vector, WT Sting , or Gt Sting . Transduced cells were transfected with 4 μg/ml of c-di-GMP or c-di-AMP for 4 h. RNA was harvested, and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. Data are representative of those from at least three independent experiments. *, P
Figure Legend Snippet: Sting is required for cyclic dinucleotide-induced IFN-β production. Sting +/+ , Sting Gt / Gt , or Sting − / − bone marrow-derived macrophages were treated with 4 μg/ml c-di-GMP (A) or c-di-AMP (B) for 4 h. RNA was harvested, and the amounts of IFN-β transcripts were measured relative to those of rps17 (A) or β-actin (B) transcripts. (C) Sting +/+ or Sting Gt / Gt immortalized bone marrow-derived macrophages were transduced with either empty MSCV2.2 vector, WT Sting , or Gt Sting . Transduced cells were transfected with 4 μg/ml of c-di-GMP or c-di-AMP for 4 h. RNA was harvested, and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. Data are representative of those from at least three independent experiments. *, P

Techniques Used: Derivative Assay, Transduction, Plasmid Preparation, Transfection

28) Product Images from "Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells"

Article Title: Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2017.00272

ERα36 overexpression enhancing M4-dependent migration but not proliferation or apoptotic escape. (A) Representative western blot of a M4 kinetic treatment of the MCF-10A cell line. M4 (1 nM) stimulated ERα36 protein expression. β-Actin was used as a loading control. N = 3. (B) Quantification of MCF-10A/ERα36 cell viability by crystal violet assay after 48 h vehicle or 1 nM M4 exposure. Histogram depicts the percentage of MCF-10A/ERα36 viable cells compared to MCF-10A/Zeo cells after treatment. M4 triggered a significant 18. 4% increase of MCF-10A/ERα36 cell proliferation rate. Each bar represents mean ± SD. N = 5. * = significantly different from MCF-10A cells. aa = significantly different from MCF-10A/ERα36 vehicle treated cells (* p
Figure Legend Snippet: ERα36 overexpression enhancing M4-dependent migration but not proliferation or apoptotic escape. (A) Representative western blot of a M4 kinetic treatment of the MCF-10A cell line. M4 (1 nM) stimulated ERα36 protein expression. β-Actin was used as a loading control. N = 3. (B) Quantification of MCF-10A/ERα36 cell viability by crystal violet assay after 48 h vehicle or 1 nM M4 exposure. Histogram depicts the percentage of MCF-10A/ERα36 viable cells compared to MCF-10A/Zeo cells after treatment. M4 triggered a significant 18. 4% increase of MCF-10A/ERα36 cell proliferation rate. Each bar represents mean ± SD. N = 5. * = significantly different from MCF-10A cells. aa = significantly different from MCF-10A/ERα36 vehicle treated cells (* p

Techniques Used: Over Expression, Migration, Western Blot, Expressing, Crystal Violet Assay

29) Product Images from "Notch1 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner"

Article Title: Notch1 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner

Journal: Molecular Cancer

doi: 10.1186/s12943-015-0295-3

Inhibition and activation of Notch signaling, respectively, in both MCF-7 and MDA-MB-231 cells. Normal group: MCF-7 or MDA-MB-231 cells were incubated under normal conditions; shNC group: MCF-7 or MDA-MB-231 cells were stably transfected with NC shRNA; shNotch1 group: MCF-7 or MDA-MB-231 cells were stably transfected with Notch1 shRNA; normal + Jagged1: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h; shNC + Jagged1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h after stable shNC transfection. Jagged1 + shNotch1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h and then Notch1 shRNA was transiently transfected for an additional 48 h. (A) , (C) , and (E) Total protein was isolated for western blot analysis using Notch1, Hey1, Hes1, and NF-κB65 antibodies. β-Actin was used as a loading control. (B) , (D) , and (F) Total RNA was extracted, and the expression levels of Notch1, Hey1, Hes1, and NF-κB65 were assayed by real-time PCR. The expression of every target gene was quantified using GAPDH as a normalization control. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P
Figure Legend Snippet: Inhibition and activation of Notch signaling, respectively, in both MCF-7 and MDA-MB-231 cells. Normal group: MCF-7 or MDA-MB-231 cells were incubated under normal conditions; shNC group: MCF-7 or MDA-MB-231 cells were stably transfected with NC shRNA; shNotch1 group: MCF-7 or MDA-MB-231 cells were stably transfected with Notch1 shRNA; normal + Jagged1: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h; shNC + Jagged1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h after stable shNC transfection. Jagged1 + shNotch1 group: MCF-7 or MDA-MB-231 cells were treated with Jagged1 for 48 h and then Notch1 shRNA was transiently transfected for an additional 48 h. (A) , (C) , and (E) Total protein was isolated for western blot analysis using Notch1, Hey1, Hes1, and NF-κB65 antibodies. β-Actin was used as a loading control. (B) , (D) , and (F) Total RNA was extracted, and the expression levels of Notch1, Hey1, Hes1, and NF-κB65 were assayed by real-time PCR. The expression of every target gene was quantified using GAPDH as a normalization control. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P

Techniques Used: Inhibition, Activation Assay, Multiple Displacement Amplification, Incubation, Stable Transfection, Transfection, shRNA, Isolation, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Characteristic of EMT under conditions of Notch1 knockdown or Jagged1 induced Notch signaling activation in breast cancer cells. (A) and (B) Morphological changes of MCF-7 and MDA-MB-231 cells. (C) Total protein was extracted for immunoblotting analysis of the EMT-related markers E-cadherin, occludin, N-cadherin, and vimentin in MCF-7 and MDA-MB-231 cells. β-Actin was used as a loading control. (D) Real-time PCR analysis of the EMT-related markers E-cadherin, occludin, N-cadherin, and vimentin in MCF-7 and MDA-MB-231 cells. GAPAH was used as a normalization control. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P
Figure Legend Snippet: Characteristic of EMT under conditions of Notch1 knockdown or Jagged1 induced Notch signaling activation in breast cancer cells. (A) and (B) Morphological changes of MCF-7 and MDA-MB-231 cells. (C) Total protein was extracted for immunoblotting analysis of the EMT-related markers E-cadherin, occludin, N-cadherin, and vimentin in MCF-7 and MDA-MB-231 cells. β-Actin was used as a loading control. (D) Real-time PCR analysis of the EMT-related markers E-cadherin, occludin, N-cadherin, and vimentin in MCF-7 and MDA-MB-231 cells. GAPAH was used as a normalization control. The data are from three independent experiments. Column: mean; bar: SD. The symbol * represents a significant difference (P

Techniques Used: Activation Assay, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction

The expression of Notch1 and Jagged1 in human breast cancer cell lines. (A) The protein expression of N1ICD in a panel of breast cancer cells (MDA-MB-231, T47D, MCF-7, ZR-75-1, and SK-BR-3), HMECs, and MCF-10A cells was evaluated by western blot. Protein samples (150 μg) were separated by 10% SDS-PAGE. β-Actin was used as a loading control. (B) The mRNA expression of Notch1 and Jagged1 was estimated by real-time PCR. GAPDH was used as a normalization control for quantifying the expression of each target gene. Experiments were performed three times. Column: mean; bar: SD.
Figure Legend Snippet: The expression of Notch1 and Jagged1 in human breast cancer cell lines. (A) The protein expression of N1ICD in a panel of breast cancer cells (MDA-MB-231, T47D, MCF-7, ZR-75-1, and SK-BR-3), HMECs, and MCF-10A cells was evaluated by western blot. Protein samples (150 μg) were separated by 10% SDS-PAGE. β-Actin was used as a loading control. (B) The mRNA expression of Notch1 and Jagged1 was estimated by real-time PCR. GAPDH was used as a normalization control for quantifying the expression of each target gene. Experiments were performed three times. Column: mean; bar: SD.

Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, SDS Page, Real-time Polymerase Chain Reaction

30) Product Images from "Loss of HuR Is Linked to Reduced Expression of Proliferative Genes during Replicative Senescence"

Article Title: Loss of HuR Is Linked to Reduced Expression of Proliferative Genes during Replicative Senescence

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.17.5889-5898.2001

Senescence-associated gene expression in WI-38 and IDH4 fibroblasts. (A) Northern blot analysis of expression of the genes indicated using early-passage (Young; ≈28 pdl) and late-passage (senescent [Sen.]; ≈60 pdl) WI-38 cells, as well as young (dex-treated) and senescent (7 days after removing dex) IDH4 cells. (B) Stabilities of cyclin A, cyclin B1, c-fos, and β-actin mRNAs in IDH4 cells that were either dex-treated [Young (+dex)] or cultured without dex for 7 days [Senescent (−dex)] were assessed after the addition of 2 μg of actinomycin D/ml; preparation of RNA at the times indicated; measurement of cyclin A, cyclin B1, c-fos, and β-actin mRNA Northern blot signals; normalizing them to 18S rRNA; and plotting them on a logarithmic scale (bottom). Dashed horizontal lines, 50% of untreated. The data represent the means ± standard errors of the means of four independent experiments.
Figure Legend Snippet: Senescence-associated gene expression in WI-38 and IDH4 fibroblasts. (A) Northern blot analysis of expression of the genes indicated using early-passage (Young; ≈28 pdl) and late-passage (senescent [Sen.]; ≈60 pdl) WI-38 cells, as well as young (dex-treated) and senescent (7 days after removing dex) IDH4 cells. (B) Stabilities of cyclin A, cyclin B1, c-fos, and β-actin mRNAs in IDH4 cells that were either dex-treated [Young (+dex)] or cultured without dex for 7 days [Senescent (−dex)] were assessed after the addition of 2 μg of actinomycin D/ml; preparation of RNA at the times indicated; measurement of cyclin A, cyclin B1, c-fos, and β-actin mRNA Northern blot signals; normalizing them to 18S rRNA; and plotting them on a logarithmic scale (bottom). Dashed horizontal lines, 50% of untreated. The data represent the means ± standard errors of the means of four independent experiments.

Techniques Used: Expressing, Northern Blot, Cell Culture

31) Product Images from "Ginkgo biloba extract EGb 761–induced upregulation of LincRNA-p21 inhibits colorectal cancer metastasis by associating with EZH2"

Article Title: Ginkgo biloba extract EGb 761–induced upregulation of LincRNA-p21 inhibits colorectal cancer metastasis by associating with EZH2

Journal: Oncotarget

doi: 10.18632/oncotarget.21345

LincRNA-p21 is associated with EZH2 in colorectal cancer (A) RIP experiments were performed using the EZH2 antibody to immunoprecipitate RNA and two primers to detect LincRNA-p21, and a relatively high enrichment of LincRNA-p21 was found compared with IgG control. (B) RIP experiments showed that no enrichment of β-actin or lncRNA control was confirmed in SW480 cells. (C) Biotinylated LincRNA-p21 or control were incubated with nuclear extracts (SW480 and SW620 cells), targeted with streptavidin beads and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and LincRNA-p21 was performed. Error bars represent median ± SD. * P
Figure Legend Snippet: LincRNA-p21 is associated with EZH2 in colorectal cancer (A) RIP experiments were performed using the EZH2 antibody to immunoprecipitate RNA and two primers to detect LincRNA-p21, and a relatively high enrichment of LincRNA-p21 was found compared with IgG control. (B) RIP experiments showed that no enrichment of β-actin or lncRNA control was confirmed in SW480 cells. (C) Biotinylated LincRNA-p21 or control were incubated with nuclear extracts (SW480 and SW620 cells), targeted with streptavidin beads and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and LincRNA-p21 was performed. Error bars represent median ± SD. * P

Techniques Used: Incubation, Western Blot

32) Product Images from "Cancer Malignancy Is Enhanced by Glyceraldehyde-Derived Advanced Glycation End-Products"

Article Title: Cancer Malignancy Is Enhanced by Glyceraldehyde-Derived Advanced Glycation End-Products

Journal: Journal of Oncology

doi: 10.1155/2010/739852

(a) and (b) Cells were incubated with control unglycated BSA or Glycer-AGEs for 24 h. The levels of TGF- β  (a) and MMP-2 (b) mRNA expression were analyzed using the real-time RT-PCR method, and the result was normalized to the  β -actin mRNA level. (c) and (d) Cells were incubated with control unglycated BSA or Glycer-AGEs for 48 h. The conditioned medium was collected, and the activities of pro-MMP-2 (c) and MMP-2 (the activated form) were measured (d). Data are shown as the mean ± SD ( n  = 3) ** P
Figure Legend Snippet: (a) and (b) Cells were incubated with control unglycated BSA or Glycer-AGEs for 24 h. The levels of TGF- β (a) and MMP-2 (b) mRNA expression were analyzed using the real-time RT-PCR method, and the result was normalized to the β -actin mRNA level. (c) and (d) Cells were incubated with control unglycated BSA or Glycer-AGEs for 48 h. The conditioned medium was collected, and the activities of pro-MMP-2 (c) and MMP-2 (the activated form) were measured (d). Data are shown as the mean ± SD ( n = 3) ** P

Techniques Used: Incubation, Expressing, Quantitative RT-PCR

RAGE expression by Western blot analysis. Cell lysates (30  μ g of proteins/lane) were loaded onto a 10% polyacrylamide gel. Size markers (kDa) are shown on the left. Equal protein loading was estimated using anti- β -actin antibody. The arrow indicates full-length RAGE.
Figure Legend Snippet: RAGE expression by Western blot analysis. Cell lysates (30  μ g of proteins/lane) were loaded onto a 10% polyacrylamide gel. Size markers (kDa) are shown on the left. Equal protein loading was estimated using anti- β -actin antibody. The arrow indicates full-length RAGE.

Techniques Used: Expressing, Western Blot

33) Product Images from "Electroacupuncture Potentiates Cannabinoid Receptor-Mediated Descending Inhibitory Control in a Mouse Model of Knee Osteoarthritis"

Article Title: Electroacupuncture Potentiates Cannabinoid Receptor-Mediated Descending Inhibitory Control in a Mouse Model of Knee Osteoarthritis

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00112

Quantitative analysis of protein and mRNA levels of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (A,C) Representative gel images show the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues obtained from control (CON), KOA, and KOA treated with 2 Hz + 1 mA EA. β-actin was used as a loading control. The protein band at 63 kDa and 45 kDa corresponds to the CB1 and CB2 receptors, respectively. (B,D) Summary data show the effect of KOA and EA on the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (E,F) Effects of KOA and EA on the mRNA level of CB1 and CB2 receptors in the midbrain. Data are expressed as means ± SD ( n = 6 mice in each group). * p
Figure Legend Snippet: Quantitative analysis of protein and mRNA levels of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (A,C) Representative gel images show the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues obtained from control (CON), KOA, and KOA treated with 2 Hz + 1 mA EA. β-actin was used as a loading control. The protein band at 63 kDa and 45 kDa corresponds to the CB1 and CB2 receptors, respectively. (B,D) Summary data show the effect of KOA and EA on the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (E,F) Effects of KOA and EA on the mRNA level of CB1 and CB2 receptors in the midbrain. Data are expressed as means ± SD ( n = 6 mice in each group). * p

Techniques Used: Mouse Assay

34) Product Images from "Inhibition of angiogenesis in endothelial cells by Human Lysyl oxidase propeptide"

Article Title: Inhibition of angiogenesis in endothelial cells by Human Lysyl oxidase propeptide

Journal: Scientific Reports

doi: 10.1038/s41598-018-28745-8

Effect of rLOX-PP on FAK (Tyr397) and ERK (Thr202/Tyr204) phosphorylation induced by VEGF in HUVECs: ( a . ( b ) Bar graph represents the quantification of western blot using ImageJ software for pFAK (Tyr397) and ( c ) pERK (Thr202/Tyr204). ( d . ( e ) Densitogram of the western blot shows the ratio of pFAK to total FAK, normalized with β-actin. ( f ) Densitogram of the western blot shows the ratio of pERK to total ERK, normalized with β-actin. Values were expressed as mean ± SD, n = 3. ** p
Figure Legend Snippet: Effect of rLOX-PP on FAK (Tyr397) and ERK (Thr202/Tyr204) phosphorylation induced by VEGF in HUVECs: ( a . ( b ) Bar graph represents the quantification of western blot using ImageJ software for pFAK (Tyr397) and ( c ) pERK (Thr202/Tyr204). ( d . ( e ) Densitogram of the western blot shows the ratio of pFAK to total FAK, normalized with β-actin. ( f ) Densitogram of the western blot shows the ratio of pERK to total ERK, normalized with β-actin. Values were expressed as mean ± SD, n = 3. ** p

Techniques Used: Western Blot, Software

Uptake and localization of rLOX-PP in HUVECs. ( a . ( b ) Densitogram of the western blot shows LOX-PP protein normalized with β-actin. ( c . ( d ) Densitogram of the western blot shows His-tagged LOX-PP protein expression normalized with β-actin. Values were expressed as mean ± SD, n = 3. ** p
Figure Legend Snippet: Uptake and localization of rLOX-PP in HUVECs. ( a . ( b ) Densitogram of the western blot shows LOX-PP protein normalized with β-actin. ( c . ( d ) Densitogram of the western blot shows His-tagged LOX-PP protein expression normalized with β-actin. Values were expressed as mean ± SD, n = 3. ** p

Techniques Used: Western Blot, Expressing

35) Product Images from "Human CD36 overexpression in renal tubules accelerates the progression of renal diseases in a mouse model of folic acid-induced acute kidney injury"

Article Title: Human CD36 overexpression in renal tubules accelerates the progression of renal diseases in a mouse model of folic acid-induced acute kidney injury

Journal: Kidney Research and Clinical Practice

doi: 10.23876/j.krcp.2018.37.1.30

Quantitative reverse transcription polymerase chain reaction-based determination of the relative mRNA expression of inflammation-, fibrosis-, and apoptosis-associated genes in the kidneys of 10-week-old mice at 2 days after folic acid administration (A) A representative image of relative Mcp1 mRNA level. Mcp1 mRNA level was significantly higher in folic acid-treated Tg mice than in vehicle-treated Tg mice. However, no significant difference was observed in Mcp1 mRNA level between folic acid-treated Tg and WT mice. (B) A representative image of relative Cd68 mRNA level. Cd68 mRNA level was significantly higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. (C) A representative image of relative Acta2 and Fn1 mRNA levels. Acta2 and Fn1 mRNA levels were higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. (D) A representative image of relative Col1a1 and Col3a1 mRNA levels. Col1a1 and Col3a1 mRNA levels were higher in folic acid-treated Tg mice than in vehicle-treated and folic acid-treated WT mice. In particular, the Col3a1 mRNA level was significantly higher in folic acid-treated Tg mice than in folic acid-treated WT mice. (E) A representative image of relative Apaf1 and Bax/Bcl2 mRNA levels. Apaf1 and Bax/Bcl2 mRNA levels were higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. Moreover, the Bax/Bcl2 mRNA level increased in folic acid-treated Tg mice. However, there was no significant difference in Bax/Bcl2 mRNA levels between folic acid-treated Tg and WT mice. Acta2 , α-smooth muscle actin; Apaf1 , apoptotic protease activating factor 1; Bax/Bcl2 , B-cell lymphoma 2-associated X/B-cell lymphoma 2; Col1a1 , collagen 1a1; Col3a1 , collagen 3a1; Fn1 , fibronectin; Mcp1 , monocyte chemoattractant protein-1; Tg, transgenic; WT, wild type. * P
Figure Legend Snippet: Quantitative reverse transcription polymerase chain reaction-based determination of the relative mRNA expression of inflammation-, fibrosis-, and apoptosis-associated genes in the kidneys of 10-week-old mice at 2 days after folic acid administration (A) A representative image of relative Mcp1 mRNA level. Mcp1 mRNA level was significantly higher in folic acid-treated Tg mice than in vehicle-treated Tg mice. However, no significant difference was observed in Mcp1 mRNA level between folic acid-treated Tg and WT mice. (B) A representative image of relative Cd68 mRNA level. Cd68 mRNA level was significantly higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. (C) A representative image of relative Acta2 and Fn1 mRNA levels. Acta2 and Fn1 mRNA levels were higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. (D) A representative image of relative Col1a1 and Col3a1 mRNA levels. Col1a1 and Col3a1 mRNA levels were higher in folic acid-treated Tg mice than in vehicle-treated and folic acid-treated WT mice. In particular, the Col3a1 mRNA level was significantly higher in folic acid-treated Tg mice than in folic acid-treated WT mice. (E) A representative image of relative Apaf1 and Bax/Bcl2 mRNA levels. Apaf1 and Bax/Bcl2 mRNA levels were higher in folic acid-treated WT and Tg mice than in vehicle-treated WT mice. Moreover, the Bax/Bcl2 mRNA level increased in folic acid-treated Tg mice. However, there was no significant difference in Bax/Bcl2 mRNA levels between folic acid-treated Tg and WT mice. Acta2 , α-smooth muscle actin; Apaf1 , apoptotic protease activating factor 1; Bax/Bcl2 , B-cell lymphoma 2-associated X/B-cell lymphoma 2; Col1a1 , collagen 1a1; Col3a1 , collagen 3a1; Fn1 , fibronectin; Mcp1 , monocyte chemoattractant protein-1; Tg, transgenic; WT, wild type. * P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Transgenic Assay

Effects of folic acid on renal histology and albuminuria (A) A representative image of PAS staining (×100) showing normal histology of the kidneys of vehicle-treated WT and Tg mice and severely dilated tubules with casts in the kidneys of folic acid-treated WT and Tg mice. (B) A representative image of PAS staining (×400) showing marked renal damage, including renal tubular epithelial cell flattening and casts in the tubular lumens, in all folic acid-treated mice. Severely dilated tubules with casts were observed in the kidneys of folic acid-treated Tg mice. (C) A representative image of immunohistochemical analysis with anti-α-SMA antibody (×400). No significant difference was observed between the kidneys of folic acid-treated WT and Tg mice. (D) The first representative image of semiquantitative scoring of tubular tissue injury showed more damage in folic acid-treated Tg mice compared with folic acid-treated WT mice and vehicle-treated Tg mice; however, the area fraction of positive α-SMA staining of α-SMA between folic acid-treated WT and Tg mice did not show a significant difference in the second representative image. (E) A representative image of quantitative data for albuminuria, as determined using Albuwell M ELISA kit (Exocell Inc., Philadelphia, PA, USA) and creatinine ELISA kit (Exocell Inc.). Albuminuria was higher in folic acid-treated Tg mice (n = 7) than in vehicle-treated WT (n = 7) and Tg mice (n = 7) and folic acid-treated WT mice (n = 7). α-SMA, mouse α-smooth muscle actin; PAS, periodic acid Schiff; Tg, transgenic; UACR, the urinary albumin to creatinine ratio; WT, wild type. * P
Figure Legend Snippet: Effects of folic acid on renal histology and albuminuria (A) A representative image of PAS staining (×100) showing normal histology of the kidneys of vehicle-treated WT and Tg mice and severely dilated tubules with casts in the kidneys of folic acid-treated WT and Tg mice. (B) A representative image of PAS staining (×400) showing marked renal damage, including renal tubular epithelial cell flattening and casts in the tubular lumens, in all folic acid-treated mice. Severely dilated tubules with casts were observed in the kidneys of folic acid-treated Tg mice. (C) A representative image of immunohistochemical analysis with anti-α-SMA antibody (×400). No significant difference was observed between the kidneys of folic acid-treated WT and Tg mice. (D) The first representative image of semiquantitative scoring of tubular tissue injury showed more damage in folic acid-treated Tg mice compared with folic acid-treated WT mice and vehicle-treated Tg mice; however, the area fraction of positive α-SMA staining of α-SMA between folic acid-treated WT and Tg mice did not show a significant difference in the second representative image. (E) A representative image of quantitative data for albuminuria, as determined using Albuwell M ELISA kit (Exocell Inc., Philadelphia, PA, USA) and creatinine ELISA kit (Exocell Inc.). Albuminuria was higher in folic acid-treated Tg mice (n = 7) than in vehicle-treated WT (n = 7) and Tg mice (n = 7) and folic acid-treated WT mice (n = 7). α-SMA, mouse α-smooth muscle actin; PAS, periodic acid Schiff; Tg, transgenic; UACR, the urinary albumin to creatinine ratio; WT, wild type. * P

Techniques Used: Staining, Mouse Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Transgenic Assay

36) Product Images from "Body mass-specific Na+-K+-ATPase activity in the medullary thick ascending limb: implications for species-dependent urine concentrating mechanisms"

Article Title: Body mass-specific Na+-K+-ATPase activity in the medullary thick ascending limb: implications for species-dependent urine concentrating mechanisms

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

doi: 10.1152/ajpregu.00289.2017

Na + -K + -ATPase α1-mRNA expression in the ISOM of the kangaroo rat compared with the Munich-Wistar rat ( A ) and kangaroo rat compared with the Sprague-Dawley rat ( B ), as determined by real-time PCR. β-Actin expression was not significantly different between the two species in each panel. Arbitrary units; means shown in parentheses. *Significant difference in gene expression between species by Student’s unpaired t -test.
Figure Legend Snippet: Na + -K + -ATPase α1-mRNA expression in the ISOM of the kangaroo rat compared with the Munich-Wistar rat ( A ) and kangaroo rat compared with the Sprague-Dawley rat ( B ), as determined by real-time PCR. β-Actin expression was not significantly different between the two species in each panel. Arbitrary units; means shown in parentheses. *Significant difference in gene expression between species by Student’s unpaired t -test.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Na + -K + -ATPase α1-protein expression in the ISOM of the kangaroo rat (KR) and the Munich-Wistar rat (MW). A : immunoblots with 20 μg of protein applied to each lane; each lane represents a different animal. B : quantification of Na + -K + -ATPase α1 immunoblot normalized to β-actin; n = 6 for each species. Kangaroo rat data are expressed relative to the mean of the Munich-Wistar rat, which was set to 1. *Significant difference in protein expression between species by Student’s unpaired t -test.
Figure Legend Snippet: Na + -K + -ATPase α1-protein expression in the ISOM of the kangaroo rat (KR) and the Munich-Wistar rat (MW). A : immunoblots with 20 μg of protein applied to each lane; each lane represents a different animal. B : quantification of Na + -K + -ATPase α1 immunoblot normalized to β-actin; n = 6 for each species. Kangaroo rat data are expressed relative to the mean of the Munich-Wistar rat, which was set to 1. *Significant difference in protein expression between species by Student’s unpaired t -test.

Techniques Used: Expressing, Western Blot

37) Product Images from "Propolin C Inhibited Migration and Invasion via Suppression of EGFR-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Cancer Cells"

Article Title: Propolin C Inhibited Migration and Invasion via Suppression of EGFR-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Cancer Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/7202548

Effects of propolin C on EGFR-mediated EMT marker expressions in HCC827 lung cancer cells . HCC827 cells were incubated with (a) propolin C (2.5, 5, 7.5, and 10  μ M) or (b) 10  μ M of propolin C (PPC) and 1 nM of ZD1839 (ZD) for 24 h. After incubation, cell lysates were harvested and Western blot analyses were performed to detect the expressions of E-cadherin, vimentin, snail, and  β -actin. Data represented at least three independent experiments. Significant difference was observed from the control group ( ∗ p
Figure Legend Snippet: Effects of propolin C on EGFR-mediated EMT marker expressions in HCC827 lung cancer cells . HCC827 cells were incubated with (a) propolin C (2.5, 5, 7.5, and 10  μ M) or (b) 10  μ M of propolin C (PPC) and 1 nM of ZD1839 (ZD) for 24 h. After incubation, cell lysates were harvested and Western blot analyses were performed to detect the expressions of E-cadherin, vimentin, snail, and β -actin. Data represented at least three independent experiments. Significant difference was observed from the control group ( ∗ p

Techniques Used: Marker, Incubation, Western Blot

38) Product Images from "Expression of the glucagon-like peptide-1 receptor and its role in regulating autophagy in endometrial cancer"

Article Title: Expression of the glucagon-like peptide-1 receptor and its role in regulating autophagy in endometrial cancer

Journal: BMC Cancer

doi: 10.1186/s12885-018-4570-8

Status of AMPK phosphorylation and marker of autophagy expression in Ishikawa endometrial cancer cells treated by liraglutide. a Ishikawa cells were treated with different concentrations of liraglutide for 96 h. The cells were harvested, and AMPK, p-AMPK, LC3-II and p62 expression was analyzed by Western blot. β-actin was used as an internal control. Representative Western blot results are shown. LC3-II is defined as the lower band in the panel. b Quantification of the p-AMPK/AMPK ratio. * denotes means significantly different from control ( p
Figure Legend Snippet: Status of AMPK phosphorylation and marker of autophagy expression in Ishikawa endometrial cancer cells treated by liraglutide. a Ishikawa cells were treated with different concentrations of liraglutide for 96 h. The cells were harvested, and AMPK, p-AMPK, LC3-II and p62 expression was analyzed by Western blot. β-actin was used as an internal control. Representative Western blot results are shown. LC3-II is defined as the lower band in the panel. b Quantification of the p-AMPK/AMPK ratio. * denotes means significantly different from control ( p

Techniques Used: Marker, Expressing, Western Blot

39) Product Images from "Differential Phosphorylation of GluN1-MAPKs in Rat Brain Reward Circuits following Long-Term Alcohol Exposure"

Article Title: Differential Phosphorylation of GluN1-MAPKs in Rat Brain Reward Circuits following Long-Term Alcohol Exposure

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054930

Effects of long-term alcohol intake on N-methyl-D-aspartate-type glutamate receptors 1 (GluN1) expression and phosphorylation (Ser897) in the nucleus accumbens (NAc), caudate putamen (CPu), amygdala (Amy), hippocampus (Hip) and the prefrontal cortex (PFC). Total-GluN1 expression was significantly increased. Obvious decreases were found in the phospho/total-GluN1 in all of the five brain regions examined. Data represent mean ± SD relative to water-drinking controls that were set as 100%. β-actin was used as a loading control. * p
Figure Legend Snippet: Effects of long-term alcohol intake on N-methyl-D-aspartate-type glutamate receptors 1 (GluN1) expression and phosphorylation (Ser897) in the nucleus accumbens (NAc), caudate putamen (CPu), amygdala (Amy), hippocampus (Hip) and the prefrontal cortex (PFC). Total-GluN1 expression was significantly increased. Obvious decreases were found in the phospho/total-GluN1 in all of the five brain regions examined. Data represent mean ± SD relative to water-drinking controls that were set as 100%. β-actin was used as a loading control. * p

Techniques Used: Expressing

Decreased extracellular signal-regulated kinase (ERK) (Thr202/Tyr204) phosphorylation was found in the nucleus accumbens (NAc), caudate putamen (CPu), amygdala (Amy), hippocampus (Hip) and the prefrontal cortex (PFC) in rats following 28 d of 6% (v/v) alcohol exposure. Ratios of phospho/total-ERK protein levels in the five brain regions were analyzed. Data were expressed as mean ± SD relative to water-drinking controls that were set as 100%. β-actin was used as loading control. * p
Figure Legend Snippet: Decreased extracellular signal-regulated kinase (ERK) (Thr202/Tyr204) phosphorylation was found in the nucleus accumbens (NAc), caudate putamen (CPu), amygdala (Amy), hippocampus (Hip) and the prefrontal cortex (PFC) in rats following 28 d of 6% (v/v) alcohol exposure. Ratios of phospho/total-ERK protein levels in the five brain regions were analyzed. Data were expressed as mean ± SD relative to water-drinking controls that were set as 100%. β-actin was used as loading control. * p

Techniques Used:

Phosphorylated c- jun N-terminal kinase (JNK) (Thr783/Tyr185) and p38 (Thr180/Tyr182) are not obviously changed in alcohol-drinking rats following 28 d of 6% (v/v) alcohol exposure. Ratios of (A) phospho/total-JNK and (B) p38 protein levels in the nucleus accumbens (NAc), caudate putamen (CPu), amygdala (Amy), hippocampus (Hip) and the prefrontal cortex (PFC) were analyzed. Data represent mean ± SD relative to water-drinking controls that were set as 100%. β-actin was used as a loading control.
Figure Legend Snippet: Phosphorylated c- jun N-terminal kinase (JNK) (Thr783/Tyr185) and p38 (Thr180/Tyr182) are not obviously changed in alcohol-drinking rats following 28 d of 6% (v/v) alcohol exposure. Ratios of (A) phospho/total-JNK and (B) p38 protein levels in the nucleus accumbens (NAc), caudate putamen (CPu), amygdala (Amy), hippocampus (Hip) and the prefrontal cortex (PFC) were analyzed. Data represent mean ± SD relative to water-drinking controls that were set as 100%. β-actin was used as a loading control.

Techniques Used:

40) Product Images from "Physiological basis for muscle stiffness and weakness in a knock‐in M1592V mouse model of hyperkalemic periodic paralysis. Physiological basis for muscle stiffness and weakness in a knock‐in M1592V mouse model of hyperkalemic periodic paralysis"

Article Title: Physiological basis for muscle stiffness and weakness in a knock‐in M1592V mouse model of hyperkalemic periodic paralysis. Physiological basis for muscle stiffness and weakness in a knock‐in M1592V mouse model of hyperkalemic periodic paralysis

Journal: Physiological Reports

doi: 10.14814/phy2.12656

The total Nav1.4 protein content reached adult level by 3 weeks of age in both wild type and HyperKPP muscles. Examples of Western Blots from (A) wild‐type FVB and (B) HyperKPP muscles. Top: NaV1.4; bottom:  β ‐actin. (C) Changes in total Nav1.4 content. For the ages of 0–4 weeks, Nav1.4 protein content was determined using all hindlimb muscles to have sufficient proteins for Western Blot. For the ages from 1‐ to 6‐month old, NaV1.4 channel content was determined only in the symptomatic EDL. Nav1.4 content was first expressed as a ratio of  β ‐actin content before being converted as a ratio of the data at 1 month. Vertical bars represent the SE of five muscles (age 0–4 weeks), five wild type, and 8–9 HyperKPP EDL (age 1–6 months). There was no significant difference between wild type and HyperKPP (ANOVA,  P >  0.05).  § Mean NaV1.4 content was significantly different from the mean content at 1 month, ANOVA, and LSD  P
Figure Legend Snippet: The total Nav1.4 protein content reached adult level by 3 weeks of age in both wild type and HyperKPP muscles. Examples of Western Blots from (A) wild‐type FVB and (B) HyperKPP muscles. Top: NaV1.4; bottom: β ‐actin. (C) Changes in total Nav1.4 content. For the ages of 0–4 weeks, Nav1.4 protein content was determined using all hindlimb muscles to have sufficient proteins for Western Blot. For the ages from 1‐ to 6‐month old, NaV1.4 channel content was determined only in the symptomatic EDL. Nav1.4 content was first expressed as a ratio of β ‐actin content before being converted as a ratio of the data at 1 month. Vertical bars represent the SE of five muscles (age 0–4 weeks), five wild type, and 8–9 HyperKPP EDL (age 1–6 months). There was no significant difference between wild type and HyperKPP (ANOVA, P >  0.05). § Mean NaV1.4 content was significantly different from the mean content at 1 month, ANOVA, and LSD P

Techniques Used: Western Blot

41) Product Images from "miR-27a-3p protects against blood–brain barrier disruption and brain injury after intracerebral hemorrhage by targeting endothelial aquaporin-11"

Article Title: miR-27a-3p protects against blood–brain barrier disruption and brain injury after intracerebral hemorrhage by targeting endothelial aquaporin-11

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.001858

miR-27a-3p directly targets the 3′ UTR of the AQP11 transcript. A and B , 24 h after ICH, the levels of AQP11 mRNA and AQP11 protein in the perihematomal tissues were determined by real-time PCR and Western blotting, respectively ( n = 6/group). β-Actin was used as an internal control. C and D , rat BMECs were isolated and transfected with miR-NC, the miR-27a-3p mimic, or the miR-27a-3p inhibitor, and the levels of AQP11 mRNA and AQP11 protein were measured 24 h after transfection. β-Actin was used as an internal control. E , sequence of AQP11_3′ UTR. F and G , alignment of miR-27a-3p with the 3′ UTR of AQP11. The miR-27a-3p binding site in the 3′ UTR of AQP11 was mutated (ACUGUGA to CUGUCCA). H , a Dual-Luciferase reporter assay was performed in BMECs to verify direct targeting of miR-27a-3p on the 3′ UTR of AQP11. The in vitro experiments were repeated three times. *, p
Figure Legend Snippet: miR-27a-3p directly targets the 3′ UTR of the AQP11 transcript. A and B , 24 h after ICH, the levels of AQP11 mRNA and AQP11 protein in the perihematomal tissues were determined by real-time PCR and Western blotting, respectively ( n = 6/group). β-Actin was used as an internal control. C and D , rat BMECs were isolated and transfected with miR-NC, the miR-27a-3p mimic, or the miR-27a-3p inhibitor, and the levels of AQP11 mRNA and AQP11 protein were measured 24 h after transfection. β-Actin was used as an internal control. E , sequence of AQP11_3′ UTR. F and G , alignment of miR-27a-3p with the 3′ UTR of AQP11. The miR-27a-3p binding site in the 3′ UTR of AQP11 was mutated (ACUGUGA to CUGUCCA). H , a Dual-Luciferase reporter assay was performed in BMECs to verify direct targeting of miR-27a-3p on the 3′ UTR of AQP11. The in vitro experiments were repeated three times. *, p

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Isolation, Transfection, Sequencing, Binding Assay, Luciferase, Reporter Assay, In Vitro

42) Product Images from "Anti-cancer effects of Kochia scoparia fruit in human breast cancer cells"

Article Title: Anti-cancer effects of Kochia scoparia fruit in human breast cancer cells

Journal: Pharmacognosy Magazine

doi: 10.4103/0973-1296.139812

Effects of MEKS on the activations of apoptosis-related proteins. MDA-MB-231 cells were treated with 0, 25, or 50 μg/ml of MEKS for 4 h or 30 nM paclitaxel for 24 h (positive control). Protein expressions of cleaved caspase 3 (C), 8 (B) and 9 (A), cleaved PARP (D) and beta-actin (the loading control) were detected by Western blotting
Figure Legend Snippet: Effects of MEKS on the activations of apoptosis-related proteins. MDA-MB-231 cells were treated with 0, 25, or 50 μg/ml of MEKS for 4 h or 30 nM paclitaxel for 24 h (positive control). Protein expressions of cleaved caspase 3 (C), 8 (B) and 9 (A), cleaved PARP (D) and beta-actin (the loading control) were detected by Western blotting

Techniques Used: Multiple Displacement Amplification, Positive Control, Western Blot

43) Product Images from "AT1 and AT2 receptors modulate renal tubular cell necroptosis in angiotensin II-infused renal injury mice"

Article Title: AT1 and AT2 receptors modulate renal tubular cell necroptosis in angiotensin II-infused renal injury mice

Journal: Scientific Reports

doi: 10.1038/s41598-019-55550-8

Inhibition of AT1R and AT2R diminishes necroptosis-related proteins in Ang II-induced renal injury mice. Tissue lysates of the retrieved mouse kidneys were subjected to SDS-PAGE and Western blotting with RIP3, p-RIP3, MLKL and p-MLKL ( A ), AT1R and AT2R ( F ) antibodies and a β-actin antibody. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 ( B–E,G,H ). For the data in ( B–E,G,H ), the values are reported as the means ± S.E.M. of n = 6; *p
Figure Legend Snippet: Inhibition of AT1R and AT2R diminishes necroptosis-related proteins in Ang II-induced renal injury mice. Tissue lysates of the retrieved mouse kidneys were subjected to SDS-PAGE and Western blotting with RIP3, p-RIP3, MLKL and p-MLKL ( A ), AT1R and AT2R ( F ) antibodies and a β-actin antibody. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 ( B–E,G,H ). For the data in ( B–E,G,H ), the values are reported as the means ± S.E.M. of n = 6; *p

Techniques Used: Inhibition, Mouse Assay, SDS Page, Western Blot

The AT2 agonist CGP42112A facilitates necroptosis of HK-2 cells. Effects of CGP42112A on HK-2 cells necroptosis were determined. HK-2 cells stimulated with CGP42112A at a concentration of 0, 0.1, 1, and 10µmol/L for 24 h. Then, qPCR and Western Blot analysis was performed to detect the level of RIP3 and MLKL mRNA and protein ( A – E ). Next, HK-2 cells were pretreated with an AT2R antagonist (10 µM PD123319) and a RIP1 inhibitor (50 µM Nec-1) for 30 min, then the cells were treated with 0.1 µM CGP42112A for 24 h. F shows representative images of immunofluorescence staining for RIP3 (red fluorescence) and in situ fluorescence TUNEL staining (green fluorescence). Scale bars represent 50 μm. ( G ) The data are presented as the % ratio of necroptotic HK-2 cells (TUNEL-positive and RIP3-positive cells). Representative blots ( H ) and Western blot analysis ( I ) for RIP3, MLKL, p-RIP3, and p-MLKL proteins; β-actin was used as a loading control. The results shown are representative of three independent experiments. *p
Figure Legend Snippet: The AT2 agonist CGP42112A facilitates necroptosis of HK-2 cells. Effects of CGP42112A on HK-2 cells necroptosis were determined. HK-2 cells stimulated with CGP42112A at a concentration of 0, 0.1, 1, and 10µmol/L for 24 h. Then, qPCR and Western Blot analysis was performed to detect the level of RIP3 and MLKL mRNA and protein ( A – E ). Next, HK-2 cells were pretreated with an AT2R antagonist (10 µM PD123319) and a RIP1 inhibitor (50 µM Nec-1) for 30 min, then the cells were treated with 0.1 µM CGP42112A for 24 h. F shows representative images of immunofluorescence staining for RIP3 (red fluorescence) and in situ fluorescence TUNEL staining (green fluorescence). Scale bars represent 50 μm. ( G ) The data are presented as the % ratio of necroptotic HK-2 cells (TUNEL-positive and RIP3-positive cells). Representative blots ( H ) and Western blot analysis ( I ) for RIP3, MLKL, p-RIP3, and p-MLKL proteins; β-actin was used as a loading control. The results shown are representative of three independent experiments. *p

Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Fluorescence, In Situ, TUNEL Assay

Inhibition of AT1R and AT2R mitigates the expression of Fas/FasL signaling molecules in Ang II-induced HK-2 cells. Effects of FasL blockade via neutralizing human Fas ligand/TNFSF6 antibody on Ang II-induced necroptosis in HK-2 cells were determined. HK-2 cells were pretreated with 3 µg/ml neutralizing human Fas ligand/TNFSF6 antibody for 2 h and exposed to 10 −9 M Ang II for 24 h. Then, Western blotting was performed to detect Fas and FasL ( A,D,E ), RIP3, MLKL, p-RIP3, and p-MLKL ( B,F–I ) levels and β-actin was used as a loading control. C shows representative images of immunofluorescence staining for RIP3 (red fluorescence) and in situ fluorescence TUNEL staining (green fluorescence). Scale bars represent 50 μm. (J) The data are presented as the % ratio of necroptotic HK-2 cells (TUNEL-positive and RIP3-positive cells). The results shown are representative of three independent experiments. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 **p
Figure Legend Snippet: Inhibition of AT1R and AT2R mitigates the expression of Fas/FasL signaling molecules in Ang II-induced HK-2 cells. Effects of FasL blockade via neutralizing human Fas ligand/TNFSF6 antibody on Ang II-induced necroptosis in HK-2 cells were determined. HK-2 cells were pretreated with 3 µg/ml neutralizing human Fas ligand/TNFSF6 antibody for 2 h and exposed to 10 −9 M Ang II for 24 h. Then, Western blotting was performed to detect Fas and FasL ( A,D,E ), RIP3, MLKL, p-RIP3, and p-MLKL ( B,F–I ) levels and β-actin was used as a loading control. C shows representative images of immunofluorescence staining for RIP3 (red fluorescence) and in situ fluorescence TUNEL staining (green fluorescence). Scale bars represent 50 μm. (J) The data are presented as the % ratio of necroptotic HK-2 cells (TUNEL-positive and RIP3-positive cells). The results shown are representative of three independent experiments. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 **p

Techniques Used: Inhibition, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, In Situ, TUNEL Assay

Ang II induces RIPK3-MLKL-mediated necroptosis in HK-2 cells. HK-2 cells were pretreated with or without 10 µM losartan and 10 µM PD123319 for 30 min or 50 µM Nec-1 for 30 min, followed by exposure to 10 −9 M Ang II for 24 h. Representative Representative blots ( A ) and Western blot analysis ( B–E ) for necroptosis marker proteins: RIP3, MLKL, p-RIP3, and p-MLKL antibodies were used; β-actin was used as a loading control. The results are representative of three independent experiments. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 **p
Figure Legend Snippet: Ang II induces RIPK3-MLKL-mediated necroptosis in HK-2 cells. HK-2 cells were pretreated with or without 10 µM losartan and 10 µM PD123319 for 30 min or 50 µM Nec-1 for 30 min, followed by exposure to 10 −9 M Ang II for 24 h. Representative Representative blots ( A ) and Western blot analysis ( B–E ) for necroptosis marker proteins: RIP3, MLKL, p-RIP3, and p-MLKL antibodies were used; β-actin was used as a loading control. The results are representative of three independent experiments. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 **p

Techniques Used: Western Blot, Marker

44) Product Images from "Evaluation of Antioxidant, Anti-cholinesterase, and Anti-inflammatory Effects of Culinary Mushroom Pleurotus pulmonarius"

Article Title: Evaluation of Antioxidant, Anti-cholinesterase, and Anti-inflammatory Effects of Culinary Mushroom Pleurotus pulmonarius

Journal: Mycobiology

doi: 10.5941/MYCO.2016.44.4.291

Inhibitory effect of Pleurotus pulmunarius methanol extract on lipopolysaccharide (LPS)-induced nitric oxide production and expression of inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. A, Nitric oxide production; B, Expression of iNOS. β-Actin was used as an internal control. Accumulated nitric oxide in the culture medium was determined by the Griess method. The values are means ± SD (n = 3). *** p ≤ 0.001 vs. LPS treated group.
Figure Legend Snippet: Inhibitory effect of Pleurotus pulmunarius methanol extract on lipopolysaccharide (LPS)-induced nitric oxide production and expression of inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. A, Nitric oxide production; B, Expression of iNOS. β-Actin was used as an internal control. Accumulated nitric oxide in the culture medium was determined by the Griess method. The values are means ± SD (n = 3). *** p ≤ 0.001 vs. LPS treated group.

Techniques Used: Expressing

45) Product Images from "Human microglia and astrocytes express cGAS-STING viral sensing components"

Article Title: Human microglia and astrocytes express cGAS-STING viral sensing components

Journal: Neuroscience letters

doi: 10.1016/j.neulet.2017.08.039

Human microglia and astrocyte cell lines and primary cells constitutively express robust levels of the viral DNA sensor cGAS. Immortalized human microglial (hμglia) and an astrocytic cell line (U87MG) or primary human microglia (1°MG) and astrocytes (1°Ast) (3 × 10 5 cell per well), were untreated (Con), treated with transfection reagent alone (L2K) or transfected with B-form DNA (BDNA; 0.01 or 0.1 μg/mL). At 24 hours post challenge, whole cell lysates were collected and analyzed for the expression of cGAS or the housekeeping gene product β-actin by immunoblot analysis. Blots shown are representatives of at least three independent experiments and in some cases sections of the blots have been repositioned for presentation purposes. The relative cGAS expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean +/− the standard error of the mean (SEM) of at least 3 independent experiments and an asterisk indicates a statistically significant difference from transfection reagent only treated cells.
Figure Legend Snippet: Human microglia and astrocyte cell lines and primary cells constitutively express robust levels of the viral DNA sensor cGAS. Immortalized human microglial (hμglia) and an astrocytic cell line (U87MG) or primary human microglia (1°MG) and astrocytes (1°Ast) (3 × 10 5 cell per well), were untreated (Con), treated with transfection reagent alone (L2K) or transfected with B-form DNA (BDNA; 0.01 or 0.1 μg/mL). At 24 hours post challenge, whole cell lysates were collected and analyzed for the expression of cGAS or the housekeeping gene product β-actin by immunoblot analysis. Blots shown are representatives of at least three independent experiments and in some cases sections of the blots have been repositioned for presentation purposes. The relative cGAS expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean +/− the standard error of the mean (SEM) of at least 3 independent experiments and an asterisk indicates a statistically significant difference from transfection reagent only treated cells.

Techniques Used: Transfection, Expressing

Human microglia and astrocyte cell lines and primary cells functionally respond to foreign intracellular double stranded DNA. Immortalized human microglial (hμglia) and an astrocytic cell line (U87MG) or primary human microglia (1°MG) and astrocytes (1°Ast) (3 × 10 5 cell per well) were treated with transfection reagent alone for 3 hrs (C) or exposed to intracellular B-form DNA (BDNA; 0.1 μg/mL) for 1, 2, or 3 hours (1, 2, 3). At these time points, whole cell lysates were collected and analyzed for the expression of phosphorylated IRF3 (pIRF3) by immunoblot analysis, and RNA was isolated and the expression of mRNA encoding IFN-β was determined by semi-quantitative RT-PCR. Expression of β-actin protein and GAPDH mRNA housekeeping gene products is shown. Results shown are representatives of at least three independent experiments and in some cases sections of the blots have been repositioned for presentation purposes.
Figure Legend Snippet: Human microglia and astrocyte cell lines and primary cells functionally respond to foreign intracellular double stranded DNA. Immortalized human microglial (hμglia) and an astrocytic cell line (U87MG) or primary human microglia (1°MG) and astrocytes (1°Ast) (3 × 10 5 cell per well) were treated with transfection reagent alone for 3 hrs (C) or exposed to intracellular B-form DNA (BDNA; 0.1 μg/mL) for 1, 2, or 3 hours (1, 2, 3). At these time points, whole cell lysates were collected and analyzed for the expression of phosphorylated IRF3 (pIRF3) by immunoblot analysis, and RNA was isolated and the expression of mRNA encoding IFN-β was determined by semi-quantitative RT-PCR. Expression of β-actin protein and GAPDH mRNA housekeeping gene products is shown. Results shown are representatives of at least three independent experiments and in some cases sections of the blots have been repositioned for presentation purposes.

Techniques Used: Transfection, Expressing, Isolation, Quantitative RT-PCR

46) Product Images from "Electroacupuncture Potentiates Cannabinoid Receptor-Mediated Descending Inhibitory Control in a Mouse Model of Knee Osteoarthritis"

Article Title: Electroacupuncture Potentiates Cannabinoid Receptor-Mediated Descending Inhibitory Control in a Mouse Model of Knee Osteoarthritis

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00112

Quantitative analysis of protein and mRNA levels of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (A,C) Representative gel images show the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues obtained from control (CON), KOA, and KOA treated with 2 Hz + 1 mA EA. β-actin was used as a loading control. The protein band at 63 kDa and 45 kDa corresponds to the CB1 and CB2 receptors, respectively. (B,D) Summary data show the effect of KOA and EA on the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (E,F) Effects of KOA and EA on the mRNA level of CB1 and CB2 receptors in the midbrain. Data are expressed as means ± SD ( n = 6 mice in each group). * p
Figure Legend Snippet: Quantitative analysis of protein and mRNA levels of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (A,C) Representative gel images show the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues obtained from control (CON), KOA, and KOA treated with 2 Hz + 1 mA EA. β-actin was used as a loading control. The protein band at 63 kDa and 45 kDa corresponds to the CB1 and CB2 receptors, respectively. (B,D) Summary data show the effect of KOA and EA on the protein level of CB1 and CB2 receptors in the midbrain, medulla and spinal cord tissues. (E,F) Effects of KOA and EA on the mRNA level of CB1 and CB2 receptors in the midbrain. Data are expressed as means ± SD ( n = 6 mice in each group). * p

Techniques Used: Mouse Assay

47) Product Images from "Tannins from Hamamelis virginiana Bark Extract: Characterization and Improvement of the Antiviral Efficacy against Influenza A Virus and Human Papillomavirus"

Article Title: Tannins from Hamamelis virginiana Bark Extract: Characterization and Improvement of the Antiviral Efficacy against Influenza A Virus and Human Papillomavirus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088062

Determination of possible cytotoxicity or unspecific host cell receptor inhibition. ( A ) Cell metabolic activity after 24 h of incubation of A549 cells with DMSO (no treatment), 2.5 µM of staurosporine, 10 µg/ml of UF-concentrate or 50 µg/ml of the remaining drugs was determined in triplicates using XTT assay. Optical density (OD) was determined at 450 nm after background (650 nm) subtraction and expressed as % of the untreated samples. ( B ) Caspase 3/7 activity after 24 h of A549 cell incubation with DMSO (no treatment), 2.5 µM of staurosporine, 10 µg/ml of UF-concentrate or 50 µg/ml of the remaining drugs was assayed in at least triplicates using detection of a luminogenic caspase 3/7 cleavage product. ( C ) A549 cells were infected in triplicates with adenovirus type 5 (MOI of 0.05) and simultaneously treated with UF-concentrate. TCID50 was determined at 24 h post infection. ( D ) Interference of the drugs with cellular TNF-α signaling. A549 cells were preincubated for 30 or 0 minutes (“Preinc.”+or −, respectively) with 50 µg/ml of bark extract (“Bark”) or UF-concentrate (“UF-c”). Then, 0 or 30 ng/ml TNF-α were and 15 minutes later, total proteins were extracted. IκB-α and the loading control β-actin were detected on a Western blot using specific primary and Cy-5 and Cy-3 labeled secondary antibodies. * significantly elevated caspase expression (p
Figure Legend Snippet: Determination of possible cytotoxicity or unspecific host cell receptor inhibition. ( A ) Cell metabolic activity after 24 h of incubation of A549 cells with DMSO (no treatment), 2.5 µM of staurosporine, 10 µg/ml of UF-concentrate or 50 µg/ml of the remaining drugs was determined in triplicates using XTT assay. Optical density (OD) was determined at 450 nm after background (650 nm) subtraction and expressed as % of the untreated samples. ( B ) Caspase 3/7 activity after 24 h of A549 cell incubation with DMSO (no treatment), 2.5 µM of staurosporine, 10 µg/ml of UF-concentrate or 50 µg/ml of the remaining drugs was assayed in at least triplicates using detection of a luminogenic caspase 3/7 cleavage product. ( C ) A549 cells were infected in triplicates with adenovirus type 5 (MOI of 0.05) and simultaneously treated with UF-concentrate. TCID50 was determined at 24 h post infection. ( D ) Interference of the drugs with cellular TNF-α signaling. A549 cells were preincubated for 30 or 0 minutes (“Preinc.”+or −, respectively) with 50 µg/ml of bark extract (“Bark”) or UF-concentrate (“UF-c”). Then, 0 or 30 ng/ml TNF-α were and 15 minutes later, total proteins were extracted. IκB-α and the loading control β-actin were detected on a Western blot using specific primary and Cy-5 and Cy-3 labeled secondary antibodies. * significantly elevated caspase expression (p

Techniques Used: Inhibition, Activity Assay, Incubation, XTT Assay, Infection, Western Blot, Labeling, Expressing

Effect on different IAV and HPV life cycle steps. ( A–B ) Step of the IAV life cycle affected. A549 cells were infected with pandemic H1N1 (MOI 0.1), and treated with 50 µg/ml of bark extract ( A ) or 10 µg/ml of UF-concentrate ( B ) starting 2 h before infection or 0, 2, 4 or 6 h after infection. TCID50s were determined 24 h post infection. ( C ) Effect on HPV attachment. HaCaT cells preincubated for 1 h with the original (black bars) or tannin-free (grey bars) extracts or DMSO and infected for 15 min with 500 HPV pseudovirions per cell were washed five times and collected in SDS sample buffer for Western blotting. Cell-bound HPV16 particles were stained with anti-L1 antibody and relative band intensities to the β-actin band were quantified densitometrically. ( D ) Effect on HPV capsid disassembly. HaCaT cells were treated with 20 µg/ml of the extracts for 1 h before HPV 16 pseudovirion infection for 7 h followed by fixation and staining with mouse anti-L1-7 antibody. L1-7 recognizes an epitope located inside of the pseudovirion capsid accessible after uncoating. Fluorescence of L1-7-positive pixels was normalized to the cell nucleus signal (Hoechst staining) and expressed as % of untreated. n.d., not detectable or TCID50
Figure Legend Snippet: Effect on different IAV and HPV life cycle steps. ( A–B ) Step of the IAV life cycle affected. A549 cells were infected with pandemic H1N1 (MOI 0.1), and treated with 50 µg/ml of bark extract ( A ) or 10 µg/ml of UF-concentrate ( B ) starting 2 h before infection or 0, 2, 4 or 6 h after infection. TCID50s were determined 24 h post infection. ( C ) Effect on HPV attachment. HaCaT cells preincubated for 1 h with the original (black bars) or tannin-free (grey bars) extracts or DMSO and infected for 15 min with 500 HPV pseudovirions per cell were washed five times and collected in SDS sample buffer for Western blotting. Cell-bound HPV16 particles were stained with anti-L1 antibody and relative band intensities to the β-actin band were quantified densitometrically. ( D ) Effect on HPV capsid disassembly. HaCaT cells were treated with 20 µg/ml of the extracts for 1 h before HPV 16 pseudovirion infection for 7 h followed by fixation and staining with mouse anti-L1-7 antibody. L1-7 recognizes an epitope located inside of the pseudovirion capsid accessible after uncoating. Fluorescence of L1-7-positive pixels was normalized to the cell nucleus signal (Hoechst staining) and expressed as % of untreated. n.d., not detectable or TCID50

Techniques Used: Infection, Western Blot, Staining, Fluorescence

48) Product Images from "MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart"

Article Title: MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00271.2011

miR-1 enhances differentiation of ES cells into cardiac myocytes. Representative photomicrographs show heart sections stained with anti-α-actin for cardiac myocytes at 10× ( A–C ) and 40× ( D–F ), donor transplanted
Figure Legend Snippet: miR-1 enhances differentiation of ES cells into cardiac myocytes. Representative photomicrographs show heart sections stained with anti-α-actin for cardiac myocytes at 10× ( A–C ) and 40× ( D–F ), donor transplanted

Techniques Used: Staining

miR-1-ES cells prevent cardiac myocyte apoptosis in the infarcted myocardium. Representative photomicrographs of section labeled with sarcomeric α-actin in green ( A ), TUNEL in red ( B ), DAPI in blue ( C ), and merged image ( D ) demonstrate apoptosis
Figure Legend Snippet: miR-1-ES cells prevent cardiac myocyte apoptosis in the infarcted myocardium. Representative photomicrographs of section labeled with sarcomeric α-actin in green ( A ), TUNEL in red ( B ), DAPI in blue ( C ), and merged image ( D ) demonstrate apoptosis

Techniques Used: Labeling, TUNEL Assay

49) Product Images from "Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells"

Article Title: Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2016.06.001

Mild temperature shift induced phosphorylation of H2AX in HeLa cells and CHO-K1 cells, and 3AB potentiated this effect. (A) HeLa cells were incubated at 37.0 °C or 40.5 °C with or without 7 mM 3AB for 24 h. Cells were subjected to Western blotting by using anti-phosphorylated H2AX, PARP1, PARG and p53 antibodies. β-Actin was used as a loading control. n.s.; nonspecific band. (B) CHO-K1 cells were incubated at 37.0 °C or 40.5 °C with or without 7 mM 3AB for 24 h. Cells were subjected to Western blotting by using anti-phosphorylated H2AX antibody. β-Actin was used as a loading control. (C) HeLa cells were grown on coverslips, incubated for 24 h at 37.0 °C or 40.5 °C with or without 7 mM 3AB, fixed, and were examined for γH2AX foci by immunofluorescence. (D) Quantification of γH2AX foci-positive HeLa cells cultured for 24 h was performed. For each point, 200 cells were analyzed. This experiment was repeated thrice and the mean number of foci was plotted. (E) Quantification of γH2AX foci-positive HeLa cells cultured for 12 h was performed. For each point, 200 cells were analyzed. This experiment was repeated thrice and the mean number of foci was plotted. (D, E) ** p
Figure Legend Snippet: Mild temperature shift induced phosphorylation of H2AX in HeLa cells and CHO-K1 cells, and 3AB potentiated this effect. (A) HeLa cells were incubated at 37.0 °C or 40.5 °C with or without 7 mM 3AB for 24 h. Cells were subjected to Western blotting by using anti-phosphorylated H2AX, PARP1, PARG and p53 antibodies. β-Actin was used as a loading control. n.s.; nonspecific band. (B) CHO-K1 cells were incubated at 37.0 °C or 40.5 °C with or without 7 mM 3AB for 24 h. Cells were subjected to Western blotting by using anti-phosphorylated H2AX antibody. β-Actin was used as a loading control. (C) HeLa cells were grown on coverslips, incubated for 24 h at 37.0 °C or 40.5 °C with or without 7 mM 3AB, fixed, and were examined for γH2AX foci by immunofluorescence. (D) Quantification of γH2AX foci-positive HeLa cells cultured for 24 h was performed. For each point, 200 cells were analyzed. This experiment was repeated thrice and the mean number of foci was plotted. (E) Quantification of γH2AX foci-positive HeLa cells cultured for 12 h was performed. For each point, 200 cells were analyzed. This experiment was repeated thrice and the mean number of foci was plotted. (D, E) ** p

Techniques Used: Incubation, Western Blot, Immunofluorescence, Cell Culture

50) Product Images from "Kif2a regulates spindle organization and cell cycle progression in meiotic oocytes"

Article Title: Kif2a regulates spindle organization and cell cycle progression in meiotic oocytes

Journal: Scientific Reports

doi: 10.1038/srep38574

Depletion of Kif2a causes abnormal spindles and misaligned chromosomes in mouse oocytes. ( A ) Western blotting results for Kif2a after siRNA injection were cropped gels. After microinjection of Kif2a or control siRNA, oocytes were incubated in M2 medium containing 100 μm IBMX for 24 h, then washed 5 times and placed in IBMX-free M2 medium for 8 h, followed by Western blotting. Relative expression of Kif2a after siRNA injection. The intensity of Kif2a/β-actin was assessed by gray level analysis. Data are presented as Means ± SEM of 3 independent experiments (*P
Figure Legend Snippet: Depletion of Kif2a causes abnormal spindles and misaligned chromosomes in mouse oocytes. ( A ) Western blotting results for Kif2a after siRNA injection were cropped gels. After microinjection of Kif2a or control siRNA, oocytes were incubated in M2 medium containing 100 μm IBMX for 24 h, then washed 5 times and placed in IBMX-free M2 medium for 8 h, followed by Western blotting. Relative expression of Kif2a after siRNA injection. The intensity of Kif2a/β-actin was assessed by gray level analysis. Data are presented as Means ± SEM of 3 independent experiments (*P

Techniques Used: Western Blot, Injection, Incubation, Expressing

Expression and subcellular localization of Kif2a during mouse oocyte meiotic maturation. ( A ) Western blotting results for expression of Kif2a were cropped gels. Oocytes were collected after culture for 0, 4, 8, 12 h, corresponding to the GV, Pro-M I, M I and M II stages, respectively. The molecular weight of Kif2a and β-actin were 110 kDa and 43 kDa, respectively. Each sample contained 200 oocytes. Full-length gels are presented in Supplementary Figure 1 . The intensity of Kif2a/β-actin was assessed by grey level analysis. ( B ) Subcellular localization of Kif2a shown by immunofluoresce nt staining and confocal microscopy. Oocytes at various stages (GV, GVBD, M I,T Iand M II) were stained with antibody against Kif2a. Green, Kif2a; red, DNA; Magnifications of the boxed regions are shown. Bar = 20 μm.
Figure Legend Snippet: Expression and subcellular localization of Kif2a during mouse oocyte meiotic maturation. ( A ) Western blotting results for expression of Kif2a were cropped gels. Oocytes were collected after culture for 0, 4, 8, 12 h, corresponding to the GV, Pro-M I, M I and M II stages, respectively. The molecular weight of Kif2a and β-actin were 110 kDa and 43 kDa, respectively. Each sample contained 200 oocytes. Full-length gels are presented in Supplementary Figure 1 . The intensity of Kif2a/β-actin was assessed by grey level analysis. ( B ) Subcellular localization of Kif2a shown by immunofluoresce nt staining and confocal microscopy. Oocytes at various stages (GV, GVBD, M I,T Iand M II) were stained with antibody against Kif2a. Green, Kif2a; red, DNA; Magnifications of the boxed regions are shown. Bar = 20 μm.

Techniques Used: Expressing, Western Blot, Molecular Weight, Staining, Confocal Microscopy

51) Product Images from "Increased miR-155 and heme oxygenase-1 expression is involved in the protective effects of formononetin in traumatic brain injury in rats"

Article Title: Increased miR-155 and heme oxygenase-1 expression is involved in the protective effects of formononetin in traumatic brain injury in rats

Journal: American Journal of Translational Research

doi:

The effects of formononetin on miR-155 and HO-1 levels in TBI rats. The RNA and protein extracted from the injured zone of TBI rats were analyzed by qRT-PCR and western blotting, respectively. A. Quantitative analysis using qRT-PCR of the expression of miR-155, normalized to GAPDH expression. B. Quantitative analysis using qRT-PCR of the expression of OH-1, normalized to GAPDH expression. C. Quantitative analysis using western blot of the expression of miR-155, normalized to β-actin expression. The results are expressed as mean ± SD. Δ P
Figure Legend Snippet: The effects of formononetin on miR-155 and HO-1 levels in TBI rats. The RNA and protein extracted from the injured zone of TBI rats were analyzed by qRT-PCR and western blotting, respectively. A. Quantitative analysis using qRT-PCR of the expression of miR-155, normalized to GAPDH expression. B. Quantitative analysis using qRT-PCR of the expression of OH-1, normalized to GAPDH expression. C. Quantitative analysis using western blot of the expression of miR-155, normalized to β-actin expression. The results are expressed as mean ± SD. Δ P

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

Formononetin down-regulated the mRNA and protein expression levels of BACH1 in the damaged brain of TBI rats. The RNA and protein extracted from the injured zone in rats 24 h after TBI were analyzed for BACH1 expression by qRT-PCR and western blot. A. The mRNA expression of BACH1 was normalized to GAPDH expression. B. The protein expression of BACH1 was normalized to β-actin. The results are expressed as mean ± SD. Δ P
Figure Legend Snippet: Formononetin down-regulated the mRNA and protein expression levels of BACH1 in the damaged brain of TBI rats. The RNA and protein extracted from the injured zone in rats 24 h after TBI were analyzed for BACH1 expression by qRT-PCR and western blot. A. The mRNA expression of BACH1 was normalized to GAPDH expression. B. The protein expression of BACH1 was normalized to β-actin. The results are expressed as mean ± SD. Δ P

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

52) Product Images from "miR 488* inhibits androgen receptor expression in prostate carcinoma cells"

Article Title: miR 488* inhibits androgen receptor expression in prostate carcinoma cells

Journal: International journal of cancer. Journal international du cancer

doi: 10.1002/ijc.25753

miR 488* downregulates AR expression. Representative western blots showing the expression of AR and β-actin in LNCaP ( a ) and C4-2B ( c ) cells treated with indicated amounts of miR 488* mimic or NC mimic for 48 hr. Mock transfected cells represent
Figure Legend Snippet: miR 488* downregulates AR expression. Representative western blots showing the expression of AR and β-actin in LNCaP ( a ) and C4-2B ( c ) cells treated with indicated amounts of miR 488* mimic or NC mimic for 48 hr. Mock transfected cells represent

Techniques Used: Expressing, Western Blot, Transfection

53) Product Images from "Microbial Changes and Host Response in F344 Rat Colon Depending on Sex and Age Following a High-Fat Diet"

Article Title: Microbial Changes and Host Response in F344 Rat Colon Depending on Sex and Age Following a High-Fat Diet

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02236

Inflammation and proliferation markers in colonic mucosa. Myeloperoxidase concentration (A) was assessed by ELISA. (B) Western blot of COX2, pro-caspase-1, caspase-1 p10, and cyclin D1 was performed with colonic mucosa samples. Band intensities of COX2 (C) , pro-caspase-1 (D) , caspase-1 p10 (E) , and cyclin D1 (F) were quantified by densitometry, adjusted to that of β-actin, an endogenous control, and normalized to the mean value of 6w.M.C group. Box and whiskers (Turkey): the central line on each box, median; the edges of boxes, 25th and 75th percentiles; whiskers, maximum and minimum values of the data. p -Values to Kruskal–Wallis test are designated on the figure; ∗ p
Figure Legend Snippet: Inflammation and proliferation markers in colonic mucosa. Myeloperoxidase concentration (A) was assessed by ELISA. (B) Western blot of COX2, pro-caspase-1, caspase-1 p10, and cyclin D1 was performed with colonic mucosa samples. Band intensities of COX2 (C) , pro-caspase-1 (D) , caspase-1 p10 (E) , and cyclin D1 (F) were quantified by densitometry, adjusted to that of β-actin, an endogenous control, and normalized to the mean value of 6w.M.C group. Box and whiskers (Turkey): the central line on each box, median; the edges of boxes, 25th and 75th percentiles; whiskers, maximum and minimum values of the data. p -Values to Kruskal–Wallis test are designated on the figure; ∗ p

Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot

54) Product Images from "Pyocyanin-induced mucin production is associated with redox modification of FOXA2"

Article Title: Pyocyanin-induced mucin production is associated with redox modification of FOXA2

Journal: Respiratory Research

doi: 10.1186/1465-9921-14-82

Restoration of FOXA2 expression by GSH attenuates PCN-mediated overexpression of MUC5AC and MUC5B mucins. (A-D) GSH treatment restores the expression of FOXA2 and attenuates the expression of MUC5AC and MUC5B mucins. NCI-H292 cells with or without GSH pretreatment were exposed to PCN (12.5 μg/ml) or sterile water (control) for 24 hr. Nuclear proteins were harvested for Western blots using anti-FOXA2 whereas total protein was probed with anti-MUC5AC or MUC5B antibodies (A) and quantified with densitometry (B-D) . The same membranes were stripped and probed with antibody nuclear protein Histone H3 for loading control for FOXA2 and β-actin as loading control for MUC5AC and MUC5B. The experiments were independently repeated three times. Densitometry data represent the mean ± SD from three experiments. * p
Figure Legend Snippet: Restoration of FOXA2 expression by GSH attenuates PCN-mediated overexpression of MUC5AC and MUC5B mucins. (A-D) GSH treatment restores the expression of FOXA2 and attenuates the expression of MUC5AC and MUC5B mucins. NCI-H292 cells with or without GSH pretreatment were exposed to PCN (12.5 μg/ml) or sterile water (control) for 24 hr. Nuclear proteins were harvested for Western blots using anti-FOXA2 whereas total protein was probed with anti-MUC5AC or MUC5B antibodies (A) and quantified with densitometry (B-D) . The same membranes were stripped and probed with antibody nuclear protein Histone H3 for loading control for FOXA2 and β-actin as loading control for MUC5AC and MUC5B. The experiments were independently repeated three times. Densitometry data represent the mean ± SD from three experiments. * p

Techniques Used: Expressing, Over Expression, Western Blot

PCN induces the expression of mucins in NCI-H292 and 16HBE airways epithelial cells. (A - D) NCI-H292 cells were exposed to PCN at indicated concentrations for 24 hr. (A - B) qRT-PCR of MUC5AC and MUC5B genes in the presence of PCN. (C - F) Western blotting and densitometry analyses of the expression of both MUC5AC (C - D) and MUC5B (E - F) . The same membranes were stripped and probed with antibody against β-actin for loading controls. (G - H) Expression of MUC5AC (G) and MUC5B (H) mucins in NCI-H292 and 16HBE cells after 24 hr of exposure to clinically relevant concentrations of PCN, and quantified by ELISA. The qRT-PCR and ELISA experiments were performed in triplicates in three independent experiments. The western blotting experiments were repeated three times with similar results. Data for qRT-PCR, ELISA and densitometry of western blots represent the mean ± SD from all three experiments. * p
Figure Legend Snippet: PCN induces the expression of mucins in NCI-H292 and 16HBE airways epithelial cells. (A - D) NCI-H292 cells were exposed to PCN at indicated concentrations for 24 hr. (A - B) qRT-PCR of MUC5AC and MUC5B genes in the presence of PCN. (C - F) Western blotting and densitometry analyses of the expression of both MUC5AC (C - D) and MUC5B (E - F) . The same membranes were stripped and probed with antibody against β-actin for loading controls. (G - H) Expression of MUC5AC (G) and MUC5B (H) mucins in NCI-H292 and 16HBE cells after 24 hr of exposure to clinically relevant concentrations of PCN, and quantified by ELISA. The qRT-PCR and ELISA experiments were performed in triplicates in three independent experiments. The western blotting experiments were repeated three times with similar results. Data for qRT-PCR, ELISA and densitometry of western blots represent the mean ± SD from all three experiments. * p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

55) Product Images from "Ethanol Extract of Evodia rutaecarpa Attenuates Cell Growth through Caspase-Dependent Apoptosis in Benign Prostatic Hyperplasia-1 Cells"

Article Title: Ethanol Extract of Evodia rutaecarpa Attenuates Cell Growth through Caspase-Dependent Apoptosis in Benign Prostatic Hyperplasia-1 Cells

Journal: Nutrients

doi: 10.3390/nu10040523

Effect of EEER on the growth of BPH-1 cells. ( A ) Cells were treated with vehicle or EEER for 24 and 48 h at the indicated concentration and the relative cell viability was assessed using the CCK-8 assay. DMSO was used as the vehicle. ( B ) The assessment of protein expression levels was performed via western blot analysis ( left ). The relative cyclin D1 and PCNA levels and phospho-ERK1/2 levels were normalized to that of β-actin and ERK1/2 levels, respectively. The densitometric analysis was performed using the Image Lab software ( right ). The data are the mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Effect of EEER on the growth of BPH-1 cells. ( A ) Cells were treated with vehicle or EEER for 24 and 48 h at the indicated concentration and the relative cell viability was assessed using the CCK-8 assay. DMSO was used as the vehicle. ( B ) The assessment of protein expression levels was performed via western blot analysis ( left ). The relative cyclin D1 and PCNA levels and phospho-ERK1/2 levels were normalized to that of β-actin and ERK1/2 levels, respectively. The densitometric analysis was performed using the Image Lab software ( right ). The data are the mean ± SEM of three independent experiments. * p

Techniques Used: Concentration Assay, CCK-8 Assay, Expressing, Western Blot, Software

56) Product Images from "Temporal downregulation of the polyubiquitin gene Ubb affects neuronal differentiation, but not maturation, in cells cultured in vitro"

Article Title: Temporal downregulation of the polyubiquitin gene Ubb affects neuronal differentiation, but not maturation, in cells cultured in vitro

Journal: Scientific Reports

doi: 10.1038/s41598-018-21032-6

Activation of Notch signaling and its consequences via Ubb KD on DIV1. ( a , d , e ) Cells isolated from embryonic brains on 14.5 dpc ( n = 3) were infected with lentivirus harboring scrambled RNA (−sh Ubb ) or Ubb shRNA (+sh Ubb ) on DIV1 and cultured for another 4 days (DIV5). Hes5 , Hey1 , NeuroD1 , and Lcn2 mRNA levels in control (−sh Ubb ) and Ubb KD (+sh Ubb ) cells were determined by qRT-PCR ( n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. ( b ) Cells were infected with lentivirus harboring scrambled RNA (−sh Ubb ) or Ubb shRNA (+sh Ubb ) on DIV1, treated with 10 μM DAPT on DIV4 to repress Notch signaling, and cultured for another 3 days (DIV7). Hes5 and Hey1 mRNA levels were determined by qRT-PCR ( n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control (−sh Ubb , −DAPT) levels. ( c ) Immunoblot detection of cleaved NICD in cells infected with lentivirus harboring scrambled RNA (−sh Ubb ) or Ubb shRNA (+sh Ubb ) on DIV1 and cultured for another 4 days (DIV5). β-Actin (β-Act) was used as a loading control. To prevent potential degradation of NICD, cells were also treated with 10 μM MG-132 for 2 hrs (+MG-132) before harvest on DIV5. ( f , g , h ) Cells isolated from embryonic brains on 14.5 dpc ( n = 3) were infected with lentivirus harboring scrambled RNA (−sh Ubb ), Ubb shRNA (+sh Ubb ), NICD1 (+NICD1), or an empty lentiviral vector (−NICD1) on DIV1 and cultured for another 4 days (DIV5). Hes5 , Hey1 , Tubb3 , and Lcn2 mRNA levels in control (−sh Ubb or −NICD1), Ubb KD (+sh Ubb ), and NICD1-overexpressing (+NICD1) cells were determined by qRT-PCR ( n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. Representative immunoblot results of cells from three different embryonic brains are shown ( c ), and data are expressed as the means ± SEM from the indicated number of samples ( a , b , d – h ). # P
Figure Legend Snippet: Activation of Notch signaling and its consequences via Ubb KD on DIV1. ( a , d , e ) Cells isolated from embryonic brains on 14.5 dpc ( n = 3) were infected with lentivirus harboring scrambled RNA (−sh Ubb ) or Ubb shRNA (+sh Ubb ) on DIV1 and cultured for another 4 days (DIV5). Hes5 , Hey1 , NeuroD1 , and Lcn2 mRNA levels in control (−sh Ubb ) and Ubb KD (+sh Ubb ) cells were determined by qRT-PCR ( n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. ( b ) Cells were infected with lentivirus harboring scrambled RNA (−sh Ubb ) or Ubb shRNA (+sh Ubb ) on DIV1, treated with 10 μM DAPT on DIV4 to repress Notch signaling, and cultured for another 3 days (DIV7). Hes5 and Hey1 mRNA levels were determined by qRT-PCR ( n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control (−sh Ubb , −DAPT) levels. ( c ) Immunoblot detection of cleaved NICD in cells infected with lentivirus harboring scrambled RNA (−sh Ubb ) or Ubb shRNA (+sh Ubb ) on DIV1 and cultured for another 4 days (DIV5). β-Actin (β-Act) was used as a loading control. To prevent potential degradation of NICD, cells were also treated with 10 μM MG-132 for 2 hrs (+MG-132) before harvest on DIV5. ( f , g , h ) Cells isolated from embryonic brains on 14.5 dpc ( n = 3) were infected with lentivirus harboring scrambled RNA (−sh Ubb ), Ubb shRNA (+sh Ubb ), NICD1 (+NICD1), or an empty lentiviral vector (−NICD1) on DIV1 and cultured for another 4 days (DIV5). Hes5 , Hey1 , Tubb3 , and Lcn2 mRNA levels in control (−sh Ubb or −NICD1), Ubb KD (+sh Ubb ), and NICD1-overexpressing (+NICD1) cells were determined by qRT-PCR ( n = 3 each), normalized against Gapdh levels, and expressed as a fold change relative to control levels. Representative immunoblot results of cells from three different embryonic brains are shown ( c ), and data are expressed as the means ± SEM from the indicated number of samples ( a , b , d – h ). # P

Techniques Used: Activation Assay, Isolation, Infection, shRNA, Cell Culture, Quantitative RT-PCR, Activated Clotting Time Assay, Plasmid Preparation

57) Product Images from "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation"

Article Title: Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation

Journal: Oncotarget

doi:

Analysis of GALNT3-mediated MUC1 glycosylation in EOC cells A. Western blot analysis of GALNT3 and MUC1 endogenous protein expression in different EOC cell lines, including A2780s; β-actin was used as a loading control; B. Semi-quantitative RT-PCR analysis of MUC1 mRNA levels in the control clone (ctrl) and shRNA- GALNT3 clones 1 and 2 (sh-G1 and sh-G2); the GUSB gene was used as an internal control; C. Western blot analysis of MUC1 expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones before (input) and following pull-down assay using biotin-conjugated VVA lectin and streptavidin agarose (pull-down); D. VVA-lectin-mediated immunoblot analysis of GalNAc-conjugated proteins in protein lysates of the ctrl, the sh-G1 and the sh-G2 A2780s clones following VVA lectin pull-down assay (pull-down). The arrow indicates bands corresponding to possible GalNAc-conjugated MUC1 peptides; E. Western blot analysis for E-cadherin and β-catenin expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones; β-actin was used as a loading control
Figure Legend Snippet: Analysis of GALNT3-mediated MUC1 glycosylation in EOC cells A. Western blot analysis of GALNT3 and MUC1 endogenous protein expression in different EOC cell lines, including A2780s; β-actin was used as a loading control; B. Semi-quantitative RT-PCR analysis of MUC1 mRNA levels in the control clone (ctrl) and shRNA- GALNT3 clones 1 and 2 (sh-G1 and sh-G2); the GUSB gene was used as an internal control; C. Western blot analysis of MUC1 expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones before (input) and following pull-down assay using biotin-conjugated VVA lectin and streptavidin agarose (pull-down); D. VVA-lectin-mediated immunoblot analysis of GalNAc-conjugated proteins in protein lysates of the ctrl, the sh-G1 and the sh-G2 A2780s clones following VVA lectin pull-down assay (pull-down). The arrow indicates bands corresponding to possible GalNAc-conjugated MUC1 peptides; E. Western blot analysis for E-cadherin and β-catenin expression in the ctrl, the sh-G1 and the sh-G2 A2780s clones; β-actin was used as a loading control

Techniques Used: Western Blot, Expressing, Quantitative RT-PCR, shRNA, Clone Assay, Pull Down Assay

58) Product Images from "Accumulation of Cav3.2 T-type Calcium Channels in the Uninjured Sural Nerve Contributes to Neuropathic Pain in Rats with Spared Nerve Injury"

Article Title: Accumulation of Cav3.2 T-type Calcium Channels in the Uninjured Sural Nerve Contributes to Neuropathic Pain in Rats with Spared Nerve Injury

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00024

Increased expression of Ca v 3.2 T-type calcium channel proteins in the uninjured sural nerve after SNI. (A) Representative Western blot bands of Ca v 3.2 T-type calcium channel proteins (molecular weight: 260 kDa) 14 days after SNI. (B) Statistical analysis of the relative band densities of Ca v 3.2 protein. β-actin is used as an internal control. Note that the expression of Ca v 3.2 T-type calcium channel protein in ipsilateral sural nerve increased in SNI rats compared with Sham-operation rats. * p
Figure Legend Snippet: Increased expression of Ca v 3.2 T-type calcium channel proteins in the uninjured sural nerve after SNI. (A) Representative Western blot bands of Ca v 3.2 T-type calcium channel proteins (molecular weight: 260 kDa) 14 days after SNI. (B) Statistical analysis of the relative band densities of Ca v 3.2 protein. β-actin is used as an internal control. Note that the expression of Ca v 3.2 T-type calcium channel protein in ipsilateral sural nerve increased in SNI rats compared with Sham-operation rats. * p

Techniques Used: Expressing, Western Blot, Molecular Weight

59) Product Images from "Inhibitory Role of Pentraxin-3 in Esophageal Squamous Cell Carcinoma"

Article Title: Inhibitory Role of Pentraxin-3 in Esophageal Squamous Cell Carcinoma

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.189921

Western blotting analysis of PTX3 expression after transfection. Expression of PTX3 in wild-type, vector-expressing, and PTX3-expressing stably transfected EC109 and KYSE-450 cells. β-actin was used as an endogenous control. PTX3: Pentraxin-3.
Figure Legend Snippet: Western blotting analysis of PTX3 expression after transfection. Expression of PTX3 in wild-type, vector-expressing, and PTX3-expressing stably transfected EC109 and KYSE-450 cells. β-actin was used as an endogenous control. PTX3: Pentraxin-3.

Techniques Used: Western Blot, Expressing, Transfection, Plasmid Preparation, Stable Transfection

60) Product Images from "5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation"

Article Title: 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation

Journal: The FASEB Journal

doi: 10.1096/fj.201500210R

Expression of 5-LOX-wt and mutants with and without FLAP in HEK293. Protein levels of 5-LOX-wt or mutants (top) and FLAP (middle) were analyzed by Western blot analysis, normalized to β-actin (bottom). Results are representative of at least 3 independent experiments.
Figure Legend Snippet: Expression of 5-LOX-wt and mutants with and without FLAP in HEK293. Protein levels of 5-LOX-wt or mutants (top) and FLAP (middle) were analyzed by Western blot analysis, normalized to β-actin (bottom). Results are representative of at least 3 independent experiments.

Techniques Used: Expressing, Western Blot

61) Product Images from "Mitofusin 2 Protects Hepatocyte Mitochondrial Function from Damage Induced by GCDCA"

Article Title: Mitofusin 2 Protects Hepatocyte Mitochondrial Function from Damage Induced by GCDCA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065455

GCDCA decreased mitochondrial fusion protein Mfn2 expression and induced mitochondria fragmentation in the treated L02 cells. (A) Representative immunoblot and quantification analysis of the protein level of Mfn2 (86 kDa) in the L02 cells. β-actin (42 kDa) was the internal standard for protein loading. (B) The specific fluorescence probe, MitoTracker Red CMXRos (red), was used to detect changes in the mitochondrial morphology. After the L02 cells were treated with various doses of GCDCA, representative changes in the mitochondrial morphology were detected under confocal microscopy. (C) Proportions of cells with fragmented mitochondrial pattern was determined in at least six different cultures under basal conditions (untreated), and after the L02 cells were treated with various doses (25, 50, 75, or 100 µM) of GCDCA for 6 h. The results are expressed as a percentage of the control value, which was set at 100%. The values are the means ± SEM. *P
Figure Legend Snippet: GCDCA decreased mitochondrial fusion protein Mfn2 expression and induced mitochondria fragmentation in the treated L02 cells. (A) Representative immunoblot and quantification analysis of the protein level of Mfn2 (86 kDa) in the L02 cells. β-actin (42 kDa) was the internal standard for protein loading. (B) The specific fluorescence probe, MitoTracker Red CMXRos (red), was used to detect changes in the mitochondrial morphology. After the L02 cells were treated with various doses of GCDCA, representative changes in the mitochondrial morphology were detected under confocal microscopy. (C) Proportions of cells with fragmented mitochondrial pattern was determined in at least six different cultures under basal conditions (untreated), and after the L02 cells were treated with various doses (25, 50, 75, or 100 µM) of GCDCA for 6 h. The results are expressed as a percentage of the control value, which was set at 100%. The values are the means ± SEM. *P

Techniques Used: Expressing, Fluorescence, Confocal Microscopy

Mitochondrial dysfunction and loss of the mitochondrial fusion protein Mfn2 in patients with extrahepatic cholestasis. (A) Representative histology of human liver tissue. (B) The MDA concentrations and (C) ATP concentrations in human liver tissue were determined using a MDA Determination Kit and an ATP Determination Kit. (D) Representative immunoblot and quantification analysis of the protein level of Mfn2 (86 kDa) in human liver tissue. β-actin (42 kDa) was the internal standard for protein loading. The results are expressed as a percentage of the control value, which was set at 100%. The values are the means ± SEM. *P
Figure Legend Snippet: Mitochondrial dysfunction and loss of the mitochondrial fusion protein Mfn2 in patients with extrahepatic cholestasis. (A) Representative histology of human liver tissue. (B) The MDA concentrations and (C) ATP concentrations in human liver tissue were determined using a MDA Determination Kit and an ATP Determination Kit. (D) Representative immunoblot and quantification analysis of the protein level of Mfn2 (86 kDa) in human liver tissue. β-actin (42 kDa) was the internal standard for protein loading. The results are expressed as a percentage of the control value, which was set at 100%. The values are the means ± SEM. *P

Techniques Used: Multiple Displacement Amplification

62) Product Images from "The protease inhibitor atazanavir blocks hERG K+ channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane"

Article Title: The protease inhibitor atazanavir blocks hERG K+ channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2014.165

Atazanavir (ATV) reduced maturation and surface expression of human ether-a-go-go -related gene ( hERG ) channels. (A) Western blot showing effects of overnight treatment with increasing concentrations of ATV (0.1, 1.0, 10, and 30 μmol/L) on hERG protein transiently expressed in HEK293 cells. (B) Image densities of fully glycosylated 155 kDa and core-glycosylated 135 kDa hERG proteins were quantified as a function of ATV concentrations using a PhosphorImager. All image densities were normalized to the β-actin protein form measured under control conditions. b P
Figure Legend Snippet: Atazanavir (ATV) reduced maturation and surface expression of human ether-a-go-go -related gene ( hERG ) channels. (A) Western blot showing effects of overnight treatment with increasing concentrations of ATV (0.1, 1.0, 10, and 30 μmol/L) on hERG protein transiently expressed in HEK293 cells. (B) Image densities of fully glycosylated 155 kDa and core-glycosylated 135 kDa hERG proteins were quantified as a function of ATV concentrations using a PhosphorImager. All image densities were normalized to the β-actin protein form measured under control conditions. b P

Techniques Used: Expressing, Western Blot

63) Product Images from "Rewarding Morphine-Induced Synaptic Function of \u03b4-Opioid Receptors on Central Glutamate Synapses"

Article Title: Rewarding Morphine-Induced Synaptic Function of \u03b4-Opioid Receptors on Central Glutamate Synapses

Journal: The Journal of Pharmacology and Experimental Therapeutics

doi: 10.1124/jpet.108.148908

Morphine increases DOR protein expression in CeA synaptosomes. A, representative lanes of Western blots of total DOR proteins and total proteins of β-actin in CeA tissues taken from a saline-treated control rat and from a morphine-treated rat.
Figure Legend Snippet: Morphine increases DOR protein expression in CeA synaptosomes. A, representative lanes of Western blots of total DOR proteins and total proteins of β-actin in CeA tissues taken from a saline-treated control rat and from a morphine-treated rat.

Techniques Used: Expressing, Western Blot

64) Product Images from "URI prevents potassium dichromate-induced oxidative stress and cell death in gastric cancer cells"

Article Title: URI prevents potassium dichromate-induced oxidative stress and cell death in gastric cancer cells

Journal: American Journal of Translational Research

doi:

URI expression in gastric cancer cells. Western blot analysis showed that URI significantly down regulated in URI siRNA transfected cells compared with the blank and scramble sequence controls. β-actin was used as an internal control.
Figure Legend Snippet: URI expression in gastric cancer cells. Western blot analysis showed that URI significantly down regulated in URI siRNA transfected cells compared with the blank and scramble sequence controls. β-actin was used as an internal control.

Techniques Used: Expressing, Western Blot, Transfection, Sequencing

65) Product Images from "miR-425 reduction causes aberrant proliferation and collagen synthesis through modulating TGF-β/Smad signaling in acute respiratory distress syndrome"

Article Title: miR-425 reduction causes aberrant proliferation and collagen synthesis through modulating TGF-β/Smad signaling in acute respiratory distress syndrome

Journal: International Journal of Clinical and Experimental Pathology

doi:

miR-425 represses KDM6A expression through targeting 3’UTR. A. Predicted interaction between miR-425 and KDM6A 3’UTR. Red letters represent the mutant nucleotides. B. Wildtype or mutant Smad2 reporter vector transfected with miR-425 mimic or inhibitor into A549 cells for 48 hours. The luciferase activities were examined using cell lysates. C. miR-425 mimic or inhibitor transiently transfected into A549 cells for 48 hours. The protein level of endogenous KDM6A was detected by immunoblotting, with the level of β-actin as a loading control. D. The segments of -2.0 kb, -0.3 kb and +0.5 kb region relative to the first code of Smad2 were quantified after ChIP assay by qPCR, 48 hours post-transfection by miR-425 mimic or inhibitor. Results were analyzed by t-test and P
Figure Legend Snippet: miR-425 represses KDM6A expression through targeting 3’UTR. A. Predicted interaction between miR-425 and KDM6A 3’UTR. Red letters represent the mutant nucleotides. B. Wildtype or mutant Smad2 reporter vector transfected with miR-425 mimic or inhibitor into A549 cells for 48 hours. The luciferase activities were examined using cell lysates. C. miR-425 mimic or inhibitor transiently transfected into A549 cells for 48 hours. The protein level of endogenous KDM6A was detected by immunoblotting, with the level of β-actin as a loading control. D. The segments of -2.0 kb, -0.3 kb and +0.5 kb region relative to the first code of Smad2 were quantified after ChIP assay by qPCR, 48 hours post-transfection by miR-425 mimic or inhibitor. Results were analyzed by t-test and P

Techniques Used: Expressing, Mutagenesis, Plasmid Preparation, Transfection, Luciferase, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

66) Product Images from "Reactive Oxygen Species-Triggered Trophoblast Apoptosis Is Initiated by Endoplasmic Reticulum Stress via Activation of Caspase-12, CHOP, and the JNK Pathway in Toxoplasma gondii Infection in Mice"

Article Title: Reactive Oxygen Species-Triggered Trophoblast Apoptosis Is Initiated by Endoplasmic Reticulum Stress via Activation of Caspase-12, CHOP, and the JNK Pathway in Toxoplasma gondii Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.06295-11

Activation of multiple proapoptotic pathways by T. gondii infection. (A) The data were normalized to the same gene, measured at GD10, in the control group. The data are means ± SDs and represent five independent experiments. Differences between gestational days were assessed by one-way ANOVA and the SNK multiple comparison posttest. (B) Placental cell lysates from different gestational days from the T. gondii infection group and GD10 from the control group were collected for immunoblotting with Abs against GRP78, CHOP, caspase-12, phospho-JNK (Thr183/Tyr185), phospho-ASK1 (Ser967), and total and phosphorylated (p-) forms of p38. β-Actin was used as an internal control. (C and D) Placental cell lysates of GD18 from three groups were checked by immunoblotting; a quantitative analysis of the Western blot analysis using densitometry (normalized to actin) is also shown. The figure is representative of five independent experiments. Values represent the mean ± SD of five samples in each group. #, P
Figure Legend Snippet: Activation of multiple proapoptotic pathways by T. gondii infection. (A) The data were normalized to the same gene, measured at GD10, in the control group. The data are means ± SDs and represent five independent experiments. Differences between gestational days were assessed by one-way ANOVA and the SNK multiple comparison posttest. (B) Placental cell lysates from different gestational days from the T. gondii infection group and GD10 from the control group were collected for immunoblotting with Abs against GRP78, CHOP, caspase-12, phospho-JNK (Thr183/Tyr185), phospho-ASK1 (Ser967), and total and phosphorylated (p-) forms of p38. β-Actin was used as an internal control. (C and D) Placental cell lysates of GD18 from three groups were checked by immunoblotting; a quantitative analysis of the Western blot analysis using densitometry (normalized to actin) is also shown. The figure is representative of five independent experiments. Values represent the mean ± SD of five samples in each group. #, P

Techniques Used: Activation Assay, Infection, Western Blot

67) Product Images from "C-reactive protein/oxidised low-density lipoprotein/?2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway"

Article Title: C-reactive protein/oxidised low-density lipoprotein/?2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway

Journal: Lipids in Health and Disease

doi: 10.1186/1476-511X-12-42

Effect of CRP / oxLDL / β2GPI on receptors associated with lipid metabolism gene expression in the aortas. After induction of diabetes and treatment with oxLDL, β2GPI, oxLDL/β2GPI complex, CRP/oxLDL/β2GPI complex and PBS (as described in the Methods section), aortas were removed and the expression of selected genes was measured using real-time PCR. Estimates of the quantity of the PCR product were obtained by densitometry using the Quantity One analysis software package. Results are show as means ± SEM of mRNA level normalized to β-actin in the normal (non-diabetes) group. A : ABCA1 mRNA and ABCG1 mRNA expression level in the aortas. B : CD36 mRNA and SR-BI mRNA expression level in the aortas. Expression level of NC group was normalized as 1, n= 4. * P
Figure Legend Snippet: Effect of CRP / oxLDL / β2GPI on receptors associated with lipid metabolism gene expression in the aortas. After induction of diabetes and treatment with oxLDL, β2GPI, oxLDL/β2GPI complex, CRP/oxLDL/β2GPI complex and PBS (as described in the Methods section), aortas were removed and the expression of selected genes was measured using real-time PCR. Estimates of the quantity of the PCR product were obtained by densitometry using the Quantity One analysis software package. Results are show as means ± SEM of mRNA level normalized to β-actin in the normal (non-diabetes) group. A : ABCA1 mRNA and ABCG1 mRNA expression level in the aortas. B : CD36 mRNA and SR-BI mRNA expression level in the aortas. Expression level of NC group was normalized as 1, n= 4. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Software

68) Product Images from "Tanshinone IIA sulfonate protects against cigarette smoke-induced COPD and down-regulation of CFTR in mice"

Article Title: Tanshinone IIA sulfonate protects against cigarette smoke-induced COPD and down-regulation of CFTR in mice

Journal: Scientific Reports

doi: 10.1038/s41598-017-18745-5

STS protects against CS-induced reduction of CFTR in 16HBE cells and mouse lungs. 16HBE cells were treated with 2% CSE for 48 h with 2 h of STS (10 μg/ml) pre-incubation. ( A ) The level of CFTR in total cell extract was determined, while β-actin used as internal control (n = 4 per group). Full-length blots are presented in supplementary Figure 2A . ( B ) Western blotting showed the impact of STS on CFTR expression in lungs from CTL, CS, CS plus STS (CS + STS) and STS-treated mice (n = 6 mice per group). Full-length blots are presented in supplementary Figure 2B . ( C ) Immunohistochemistry showed the impact of STS on CFTR expression in lungs from CTL, CS, CS plus STS (CS + STS) and STS-treated mice (n = 3 mice per group). *P
Figure Legend Snippet: STS protects against CS-induced reduction of CFTR in 16HBE cells and mouse lungs. 16HBE cells were treated with 2% CSE for 48 h with 2 h of STS (10 μg/ml) pre-incubation. ( A ) The level of CFTR in total cell extract was determined, while β-actin used as internal control (n = 4 per group). Full-length blots are presented in supplementary Figure 2A . ( B ) Western blotting showed the impact of STS on CFTR expression in lungs from CTL, CS, CS plus STS (CS + STS) and STS-treated mice (n = 6 mice per group). Full-length blots are presented in supplementary Figure 2B . ( C ) Immunohistochemistry showed the impact of STS on CFTR expression in lungs from CTL, CS, CS plus STS (CS + STS) and STS-treated mice (n = 3 mice per group). *P

Techniques Used: Incubation, Western Blot, Expressing, CTL Assay, Mouse Assay, Immunohistochemistry

69) Product Images from "HBx-induced S100A9 in NF-κB dependent manner promotes growth and metastasis of hepatocellular carcinoma cells"

Article Title: HBx-induced S100A9 in NF-κB dependent manner promotes growth and metastasis of hepatocellular carcinoma cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0512-2

Regulation of S100A9 expression by HBx-mediated NF-κB signaling activation. a , b Western blot analysis for p-NF-κB p65 levels in HepG2 cells transfected with pcDNA3.1-HBV ( a ) or pcDNA3.1-HBx ( b ) and its control pcDNA3.1 for 48 h. Total NF-κB p65 and β-actin were included as the loading controls. The densitometric ratios were compared to the controls and then normalized to the β-actin and were shown on the right panel, respectively. c Dual luciferase reporter assay analysis for transcriptional activity of NF-κB in HepG2 cells co-transfected with pcDNA3.1 or pcDNA3.1-HBV or pcDNA3.1-HBx and luciferase reporter vector (p-Luc-NF-κB). Plasmid RL-TK was also co-transfected to normalize transfection efficiency. Luciferase activity was measured 48 h after transfection. d , e HepG2 cells were transfected with pcDNA3.1-HBV ( d ) or pcDNA3.1-HBx ( e ) followed by treatment with NF-κB inhibitor BAY 11-7082 (5 μM) for 48 h. The protein levels of S100A9 were analyzed by western blot. The relative expression of S100A9 is quantified by densitometric ratios and is shown below. f , g HepG2 cells were transfected with pcDNA3.1-HBV ( f ) or pcDNA3.1-HBx ( g ) followed by treatment with siNF-κB p65 for 48 h. The protein levels of S100A9 were analyzed by western blot and quantified by densitometric ratios. h Immunofluorescence staining for p-NF-κB p65 and S100A9 in HepG2 cells transfected with pcDNA3.1-HBx followed by treatment with BAY 11-7082 (5 μM) or siNF-κB p65 for 48 h. White scale bars = 10 μm. i Western blot analysis of S100A9 in HepG2 cells transfected with pSV40-NF-κB p65 and its control pSV40 for 48 h. β-Actin was included as the loading control. The densitometric ratios were compared to the controls and then normalized to the β-actin and were shown on the right panel; * p
Figure Legend Snippet: Regulation of S100A9 expression by HBx-mediated NF-κB signaling activation. a , b Western blot analysis for p-NF-κB p65 levels in HepG2 cells transfected with pcDNA3.1-HBV ( a ) or pcDNA3.1-HBx ( b ) and its control pcDNA3.1 for 48 h. Total NF-κB p65 and β-actin were included as the loading controls. The densitometric ratios were compared to the controls and then normalized to the β-actin and were shown on the right panel, respectively. c Dual luciferase reporter assay analysis for transcriptional activity of NF-κB in HepG2 cells co-transfected with pcDNA3.1 or pcDNA3.1-HBV or pcDNA3.1-HBx and luciferase reporter vector (p-Luc-NF-κB). Plasmid RL-TK was also co-transfected to normalize transfection efficiency. Luciferase activity was measured 48 h after transfection. d , e HepG2 cells were transfected with pcDNA3.1-HBV ( d ) or pcDNA3.1-HBx ( e ) followed by treatment with NF-κB inhibitor BAY 11-7082 (5 μM) for 48 h. The protein levels of S100A9 were analyzed by western blot. The relative expression of S100A9 is quantified by densitometric ratios and is shown below. f , g HepG2 cells were transfected with pcDNA3.1-HBV ( f ) or pcDNA3.1-HBx ( g ) followed by treatment with siNF-κB p65 for 48 h. The protein levels of S100A9 were analyzed by western blot and quantified by densitometric ratios. h Immunofluorescence staining for p-NF-κB p65 and S100A9 in HepG2 cells transfected with pcDNA3.1-HBx followed by treatment with BAY 11-7082 (5 μM) or siNF-κB p65 for 48 h. White scale bars = 10 μm. i Western blot analysis of S100A9 in HepG2 cells transfected with pSV40-NF-κB p65 and its control pSV40 for 48 h. β-Actin was included as the loading control. The densitometric ratios were compared to the controls and then normalized to the β-actin and were shown on the right panel; * p

Techniques Used: Expressing, Activation Assay, Western Blot, Transfection, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Immunofluorescence, Staining

70) Product Images from "The role of LASS2 in regulating bladder cancer cell tumorigenicity in a nude mouse model"

Article Title: The role of LASS2 in regulating bladder cancer cell tumorigenicity in a nude mouse model

Journal: Oncology Letters

doi: 10.3892/ol.2017.6880

Expression of LASS2 in EJ-M3 cells following transfection. (A) EJ-M3 cells were transfected with a LASS2 overexpression plasmid or a LASS2 shRNA construct. Fluorescence images were obtained at 72 h following transfection (magnification, ×100). (B) LASS2 relative mRNA expression levels were determined by RT-qPCR analysis following transfection. (C) Western blot analysis of LASS2 protein levels following transfection. β-Actin was used as the internal control. (D) LASS2 protein levels relative to β-actin were quantified using ImageJ software. Non-infected EJ-M3 cells were used as the Con group. *P
Figure Legend Snippet: Expression of LASS2 in EJ-M3 cells following transfection. (A) EJ-M3 cells were transfected with a LASS2 overexpression plasmid or a LASS2 shRNA construct. Fluorescence images were obtained at 72 h following transfection (magnification, ×100). (B) LASS2 relative mRNA expression levels were determined by RT-qPCR analysis following transfection. (C) Western blot analysis of LASS2 protein levels following transfection. β-Actin was used as the internal control. (D) LASS2 protein levels relative to β-actin were quantified using ImageJ software. Non-infected EJ-M3 cells were used as the Con group. *P

Techniques Used: Expressing, Transfection, Over Expression, Plasmid Preparation, shRNA, Construct, Fluorescence, Quantitative RT-PCR, Western Blot, Software, Infection

71) Product Images from "MECP2 expression in gastric cancer and its correlation with clinical pathological parameters"

Article Title: MECP2 expression in gastric cancer and its correlation with clinical pathological parameters

Journal: Medicine

doi: 10.1097/MD.0000000000007691

Expression of MECP2 protein. (A) MECP2 protein expression was analyzed in GC and paired paracancerous tissues by Western blot. Expression of β-actin was used as internal control. Representative images from 2 cases were shown. (B) MECP2 expression was analyzed in GC and paired paracancerous tissues by immunochemistry. Representative images from 2 cases were shown. MECP2 was stained as brown granules. Original magnification, ×400. GC = gastric cancer, MECP2 = methyl CpG binding protein 2.
Figure Legend Snippet: Expression of MECP2 protein. (A) MECP2 protein expression was analyzed in GC and paired paracancerous tissues by Western blot. Expression of β-actin was used as internal control. Representative images from 2 cases were shown. (B) MECP2 expression was analyzed in GC and paired paracancerous tissues by immunochemistry. Representative images from 2 cases were shown. MECP2 was stained as brown granules. Original magnification, ×400. GC = gastric cancer, MECP2 = methyl CpG binding protein 2.

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

72) Product Images from "Proteomic Analysis of Chikungunya Virus Infected Microgial Cells"

Article Title: Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034800

Analysis of CHIKV infected CHME-5 cells. CHME-5 cells either mock infected or infected with CHIKV at MOI 0.1 were collected at day 2 p.i. (A, B, D) or on days 1 to 3 p.i. (C) and subsequently (A) stained with an anti-alphavirus antibody and the percentage of infected cells analyzed by flow cytometry or (B) stained with Annexin V-FITC and PI and the percentage of apoptotic cells analyzed by flow cytometery or (C, D) used for total protein extraction and (C) analyzed by western blotting with an anti-alphavirus monoclonal antibody and an anti-actin polyclonal antibody or (D) the differential proteome determined by 2D-PAGE. Representative gels from 6 biological replicates are shown. (A and B) Bar graphs represent the means ± SD of 6 replications.
Figure Legend Snippet: Analysis of CHIKV infected CHME-5 cells. CHME-5 cells either mock infected or infected with CHIKV at MOI 0.1 were collected at day 2 p.i. (A, B, D) or on days 1 to 3 p.i. (C) and subsequently (A) stained with an anti-alphavirus antibody and the percentage of infected cells analyzed by flow cytometry or (B) stained with Annexin V-FITC and PI and the percentage of apoptotic cells analyzed by flow cytometery or (C, D) used for total protein extraction and (C) analyzed by western blotting with an anti-alphavirus monoclonal antibody and an anti-actin polyclonal antibody or (D) the differential proteome determined by 2D-PAGE. Representative gels from 6 biological replicates are shown. (A and B) Bar graphs represent the means ± SD of 6 replications.

Techniques Used: Infection, Staining, Flow Cytometry, Cytometry, Protein Extraction, Western Blot, Polyacrylamide Gel Electrophoresis

73) Product Images from "Clofazimine Modulates the Expression of Lipid Metabolism Proteins in Mycobacterium leprae-Infected Macrophages"

Article Title: Clofazimine Modulates the Expression of Lipid Metabolism Proteins in Mycobacterium leprae-Infected Macrophages

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001936

Clofazimine counteracts M. leprae to modulate ADRP and HSL expression levels. THP-1 cells were cultured in six-well plates and infected with M. leprae (MOI = 10) for 24 h. Clofazimine (2.0 µg/ml) was added and incubation continued another 24 and 48 h (48 and 72 h from M. leprae infection). Total RNA and total cellular protein were purified and RT-PCR and Western blot analyses of ADRP, HSL and β-actin were performed. Representative results from three independent experiments are shown.
Figure Legend Snippet: Clofazimine counteracts M. leprae to modulate ADRP and HSL expression levels. THP-1 cells were cultured in six-well plates and infected with M. leprae (MOI = 10) for 24 h. Clofazimine (2.0 µg/ml) was added and incubation continued another 24 and 48 h (48 and 72 h from M. leprae infection). Total RNA and total cellular protein were purified and RT-PCR and Western blot analyses of ADRP, HSL and β-actin were performed. Representative results from three independent experiments are shown.

Techniques Used: Expressing, Cell Culture, Infection, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction, Western Blot

Expression of ADRP and HSL is modulated by clofazimine in THP-1 cells infected with M. leprae . THP-1 cells were cultured in six-well plates with culture medium containing either 8.0 µg/ml rifampicin, 5.0 µg/ml dapsone or 2.0 µg/ml clofazimine with M. leprae infection (MOI = 10). After incubating for 24 h, total RNA was purified and RT-PCR analysis of ADRP, HSL and β-actin was performed. Representative results from three independent experiments are shown.
Figure Legend Snippet: Expression of ADRP and HSL is modulated by clofazimine in THP-1 cells infected with M. leprae . THP-1 cells were cultured in six-well plates with culture medium containing either 8.0 µg/ml rifampicin, 5.0 µg/ml dapsone or 2.0 µg/ml clofazimine with M. leprae infection (MOI = 10). After incubating for 24 h, total RNA was purified and RT-PCR analysis of ADRP, HSL and β-actin was performed. Representative results from three independent experiments are shown.

Techniques Used: Expressing, Infection, Cell Culture, Purification, Reverse Transcription Polymerase Chain Reaction

Only M. leprae -infected THP-1 cells are susceptible to clofazimine. THP-1 cells were cultured in six-well plates with or without 2.0 µg/ml clofazimine in the presence or absence of M. leprae infection (MOI = 10). After incubating for the indicated period of time, total RNA and total cellular protein were purified and RT-PCR and Western blot analyses of ADRP, HSL and β-actin were performed. Representative results from three independent experiments are shown.
Figure Legend Snippet: Only M. leprae -infected THP-1 cells are susceptible to clofazimine. THP-1 cells were cultured in six-well plates with or without 2.0 µg/ml clofazimine in the presence or absence of M. leprae infection (MOI = 10). After incubating for the indicated period of time, total RNA and total cellular protein were purified and RT-PCR and Western blot analyses of ADRP, HSL and β-actin were performed. Representative results from three independent experiments are shown.

Techniques Used: Infection, Cell Culture, Purification, Reverse Transcription Polymerase Chain Reaction, Western Blot

Detection of ADRP and HSL mRNA in slit-skin smear samples from leprosy patients. Total RNA was isolated from slit-skin smear specimens taken from ten BL and four LL patients (A) or from one patient before and after treatment (B). Total RNA was purified and RT-PCR analysis of ADRP, HSL and β-actin was performed. Representative results from three independent experiments are shown.
Figure Legend Snippet: Detection of ADRP and HSL mRNA in slit-skin smear samples from leprosy patients. Total RNA was isolated from slit-skin smear specimens taken from ten BL and four LL patients (A) or from one patient before and after treatment (B). Total RNA was purified and RT-PCR analysis of ADRP, HSL and β-actin was performed. Representative results from three independent experiments are shown.

Techniques Used: Isolation, Purification, Reverse Transcription Polymerase Chain Reaction

74) Product Images from "Expression of the glucagon-like peptide-1 receptor and its role in regulating autophagy in endometrial cancer"

Article Title: Expression of the glucagon-like peptide-1 receptor and its role in regulating autophagy in endometrial cancer

Journal: BMC Cancer

doi: 10.1186/s12885-018-4570-8

Status of AMPK phosphorylation and marker of autophagy expression in Ishikawa endometrial cancer cells treated by liraglutide. a Ishikawa cells were treated with different concentrations of liraglutide for 96 h. The cells were harvested, and AMPK, p-AMPK, LC3-II and p62 expression was analyzed by Western blot. β-actin was used as an internal control. Representative Western blot results are shown. LC3-II is defined as the lower band in the panel. b Quantification of the p-AMPK/AMPK ratio. * denotes means significantly different from control ( p
Figure Legend Snippet: Status of AMPK phosphorylation and marker of autophagy expression in Ishikawa endometrial cancer cells treated by liraglutide. a Ishikawa cells were treated with different concentrations of liraglutide for 96 h. The cells were harvested, and AMPK, p-AMPK, LC3-II and p62 expression was analyzed by Western blot. β-actin was used as an internal control. Representative Western blot results are shown. LC3-II is defined as the lower band in the panel. b Quantification of the p-AMPK/AMPK ratio. * denotes means significantly different from control ( p

Techniques Used: Marker, Expressing, Western Blot

75) Product Images from "Novel Omega-3 Fatty Acid Epoxygenase Metabolite Reduces Kidney Fibrosis"

Article Title: Novel Omega-3 Fatty Acid Epoxygenase Metabolite Reduces Kidney Fibrosis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17050751

In the kidney of UUO mice mRNA expression mesenchymal marker fibroblast specific protein -1 (FSP-1) ( A ) and desmin ( B ) are reduced by 19,20-EDP treatment. 19,20-EDP treatment also reduced kidney expression of the mesenchymal marker α-smooth muscle action (α-SMA) ( C ); representative photomicrographs (200×) showing α-SMA-immunopositive areas (black arrows) in the kidney of different experimental groups ( D ). All data are expressed as Mean ± SEM, * p
Figure Legend Snippet: In the kidney of UUO mice mRNA expression mesenchymal marker fibroblast specific protein -1 (FSP-1) ( A ) and desmin ( B ) are reduced by 19,20-EDP treatment. 19,20-EDP treatment also reduced kidney expression of the mesenchymal marker α-smooth muscle action (α-SMA) ( C ); representative photomicrographs (200×) showing α-SMA-immunopositive areas (black arrows) in the kidney of different experimental groups ( D ). All data are expressed as Mean ± SEM, * p

Techniques Used: Mouse Assay, Expressing, Marker

Related Articles

Positive Control:

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Nucleic Acid Electrophoresis:

Article Title: Notch1 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner
Article Snippet: Equal amounts of protein (150 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Roche). .. Antibodies were purchased from the following sources: anti-Notch1 antibody (Cell Signaling Technology, Boston, MA, USA), anti-NF-κB65 antibody (Cell Signaling Technology), anti-Hey1 antibody (Abcam, Cambridge, MA, USA), anti-Hes1 antibody (Cell Signaling Technology), anti-E-cadherin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Abcam), anti-vimentin antibody (Cell Signaling Technology), anti-occludin antibody (Proteintech Group Inc., USA), anti-Slug antibody (Abcam), anti-β-catenin antibody (Cell Signaling Technology), anti-Snail (Proteintech Group Inc.), anti-Twist antibody (Proteintech Group Inc.), anti-ZEB1 (Cell Signaling Technology), anti-ZEB2 (Abcam), anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LaminB1 antibody (Santa Cruz Biotechnology), anti-N2ICD (Santa Cruz Biotechnology), anti-N3ICD (Santa Cruz Biotechnology), and anti-N4ICD (Santa Cruz Biotechnology).

Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells
Article Snippet: An equal amount of proteins from control and each treatment sample were loaded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (10% or 15%) and transferred onto polyvinyl difluoride membranes (Millipore, Billerica, MA, USA). .. Nonspecific binding proteins were blocked with 3% skim milk in 1× phosphate-buffered saline with 0.05% Tween 20 for 60 min. Membranes were incubated with primary antibodies against antihuman IL-1β antibody (AF-201-NA, R & D Systems, Minneapolis, MN, USA), NF-κB sampler kit antibodies (#9936; Cell Signaling Technology; Danvers, MA, USA), and CEBP Antibody Sampler Kit (#12814, Cell Signaling Technology), PI3 Kinase Antibody Sampler Kit (#9655, Cell Signaling Technology), or anti-β-actin antibody (sc4778, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C.

Construct:

Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *
Article Snippet: Other antibodies used included horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen), horseradish peroxidase-conjugated anti-HA antibody (Roche Applied Science), polyclonal anti-actin antibody (clone C-11; Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Pierce). .. The ratio between these proviral constructs and the APOBEC expression vectors was normally kept at 1:4.

Electrophoresis:

Article Title: Ginkgo biloba extract EGb 761–induced upregulation of LincRNA-p21 inhibits colorectal cancer metastasis by associating with EZH2
Article Snippet: Western blot and antibodies A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). .. The primary antibodies used for western blotting were rabbit rabbit anti-human Fibronectin antibody (#sc-8422, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-human β-actin antibody (#sc-47778, 1:1000; Santa Cruz Biotechnology).

Microarray:

Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma
Article Snippet: Paragraph title: 2.2. Tissue Microarray Analysis to Detect Gremlin in Pituitary Adenoma Tissue ... Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β -actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA).

Incubation:

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *
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Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma
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Article Title: Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition
Article Snippet: .. The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with anti-ATR antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-Chk1 antibody(Santa Cruz Biotechnologies), anti-pChk1 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-ATM antibody (Abcam, Cambridge, UK) and anti-β -actin antibody (Santa Cruz Biotechnologies) for 2 h. After 1-h incubation with goat-anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnologies), the protein bands were detected with luminal reagent (GE Healthcare) and their relative intensities were quantified using Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA). .. Flow cytometry assay Cells were fixed in −20 °C pre-chilled 70% ethanol overnight, and then stained with propidium iodide.

Article Title: GPC1 exosome and its regulatory mi RNAs are specific markers for the detection and target therapy of colorectal cancer
Article Snippet: .. The membrane was blocked with 5% non‐fat dried milk for 1 hr and incubated overnight at 4°C with anti‐GPC1 antibody, anti‐CD63 antibody and anti‐β‐Actin Antibody (Santa Cruz), followed by incubation with horseradish peroxidase‐conjugated goat anti‐rabbit IgG (Cell Signaling, Danvers, MA, USA) secondary antibody for 2 hrs at RT. .. A chemiluminescence substrate (ECL Prime; Amersham, Little Chalfont, UK) was used to visualize the immunoreactive proteins.

Article Title: Notch1 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner
Article Snippet: The immunoblots were incubated in 5% (w/v) skim milk powder dissolved in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature. .. Antibodies were purchased from the following sources: anti-Notch1 antibody (Cell Signaling Technology, Boston, MA, USA), anti-NF-κB65 antibody (Cell Signaling Technology), anti-Hey1 antibody (Abcam, Cambridge, MA, USA), anti-Hes1 antibody (Cell Signaling Technology), anti-E-cadherin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Abcam), anti-vimentin antibody (Cell Signaling Technology), anti-occludin antibody (Proteintech Group Inc., USA), anti-Slug antibody (Abcam), anti-β-catenin antibody (Cell Signaling Technology), anti-Snail (Proteintech Group Inc.), anti-Twist antibody (Proteintech Group Inc.), anti-ZEB1 (Cell Signaling Technology), anti-ZEB2 (Abcam), anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LaminB1 antibody (Santa Cruz Biotechnology), anti-N2ICD (Santa Cruz Biotechnology), anti-N3ICD (Santa Cruz Biotechnology), and anti-N4ICD (Santa Cruz Biotechnology).

Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells
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Expressing:

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Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *
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Article Title: Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells
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Modification:

Article Title: Fibronectin expression is upregulated by PI-3K/Akt activation in tamoxifen-resistant breast cancer cells
Article Snippet: Reagents Dulbecco’s modified Eagle’s medium (DMEM) and phenol red-free DMEM were purchased from Thermo Scientific (Hemel Hempstead, UK). .. AKT IV, secondary HRP-conjugated antibodies, and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Western Blot:

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Article Snippet: Paragraph title: Western Blot ... An appropriate dilution of mouse anti-MG-AGE (Arg-Pyrimidine, AP) mAb (Antibodies-online, GmbH) and anti-β-actin mAb (Santa Cruz Biotechnology) were used.

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *
Article Snippet: Paragraph title: Western Blot Analysis ... The anti-β-actin antibody (sc-47778, 1:5,000) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Article Title: Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition
Article Snippet: Paragraph title: Western blots ... The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with anti-ATR antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-Chk1 antibody(Santa Cruz Biotechnologies), anti-pChk1 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-ATM antibody (Abcam, Cambridge, UK) and anti-β -actin antibody (Santa Cruz Biotechnologies) for 2 h. After 1-h incubation with goat-anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnologies), the protein bands were detected with luminal reagent (GE Healthcare) and their relative intensities were quantified using Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA).

Article Title: Ginkgo biloba extract EGb 761–induced upregulation of LincRNA-p21 inhibits colorectal cancer metastasis by associating with EZH2
Article Snippet: .. The primary antibodies used for western blotting were rabbit rabbit anti-human Fibronectin antibody (#sc-8422, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-human β-actin antibody (#sc-47778, 1:1000; Santa Cruz Biotechnology). .. Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5000; Santa Cruz Biotechnology) were used as the secondary antibodies.

Article Title: GPC1 exosome and its regulatory mi RNAs are specific markers for the detection and target therapy of colorectal cancer
Article Snippet: Paragraph title: Western blot analysis ... The membrane was blocked with 5% non‐fat dried milk for 1 hr and incubated overnight at 4°C with anti‐GPC1 antibody, anti‐CD63 antibody and anti‐β‐Actin Antibody (Santa Cruz), followed by incubation with horseradish peroxidase‐conjugated goat anti‐rabbit IgG (Cell Signaling, Danvers, MA, USA) secondary antibody for 2 hrs at RT.

Article Title: Notch1 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner
Article Snippet: Paragraph title: Western blot analysis and antibodies ... Antibodies were purchased from the following sources: anti-Notch1 antibody (Cell Signaling Technology, Boston, MA, USA), anti-NF-κB65 antibody (Cell Signaling Technology), anti-Hey1 antibody (Abcam, Cambridge, MA, USA), anti-Hes1 antibody (Cell Signaling Technology), anti-E-cadherin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Abcam), anti-vimentin antibody (Cell Signaling Technology), anti-occludin antibody (Proteintech Group Inc., USA), anti-Slug antibody (Abcam), anti-β-catenin antibody (Cell Signaling Technology), anti-Snail (Proteintech Group Inc.), anti-Twist antibody (Proteintech Group Inc.), anti-ZEB1 (Cell Signaling Technology), anti-ZEB2 (Abcam), anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LaminB1 antibody (Santa Cruz Biotechnology), anti-N2ICD (Santa Cruz Biotechnology), anti-N3ICD (Santa Cruz Biotechnology), and anti-N4ICD (Santa Cruz Biotechnology).

Article Title: Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells
Article Snippet: Paragraph title: Western Immunoblotting ... The anti-β-Actin antibody (sc1615, Santa Cruz Biotechnology) or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GTX100118, Genetex) were used as a loading controls.

Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells
Article Snippet: Paragraph title: Western blotting analysis ... Nonspecific binding proteins were blocked with 3% skim milk in 1× phosphate-buffered saline with 0.05% Tween 20 for 60 min. Membranes were incubated with primary antibodies against antihuman IL-1β antibody (AF-201-NA, R & D Systems, Minneapolis, MN, USA), NF-κB sampler kit antibodies (#9936; Cell Signaling Technology; Danvers, MA, USA), and CEBP Antibody Sampler Kit (#12814, Cell Signaling Technology), PI3 Kinase Antibody Sampler Kit (#9655, Cell Signaling Technology), or anti-β-actin antibody (sc4778, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C.

Article Title: The N-Ethyl-N-Nitrosourea-Induced Goldenticket Mouse Mutant Reveals an Essential Function of Sting in the In Vivo Interferon Response to Listeria monocytogenes and Cyclic Dinucleotides ▿
Article Snippet: Paragraph title: Western blotting. ... Overexpressed wild-type or Gt Sting was detected 24 h posttransfection using anti-hemagglutinin (anti-HA) antibody (Roche, Indianapolis, IN), and β-actin was detected using an anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Transfection:

Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *
Article Snippet: Other antibodies used included horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen), horseradish peroxidase-conjugated anti-HA antibody (Roche Applied Science), polyclonal anti-actin antibody (clone C-11; Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Pierce). .. Viral Production and Infectivity Assay —HIV-1 virions were produced from 293T cells by standard calcium phosphate transfection.

TCA Precipitation:

Article Title: Shotgun proteomics deciphered age/division of labor-related functional specification of three honeybee (Apis mellifera L.) exocrine glands
Article Snippet: Two lots of independent samples of PcGs, TGs and MGs, dissected from 10 nurse bees and 10 foragers, were homogenized and lysed in the aforementioned lysis solution, subjected to TCA precipitation, and then re-lysed in SDS-sample buffer (150 mM Tris-HCl, pH 6.8, containing 1.2% SDS and 30% glycerol). .. Immunoblotting analyses were performed using anti-IDGF4 antiserum [ ], anti-human acetyl-CoA acyltransferase 2 rabbit polyclonal antibody (Sigma-Aldrich), anti-human aldolase rabbit polyclonal antibody (Cell Signaling Technology), and anti-50kDa protein (also known as MRJP2) antiserum [ ], and anti-beta-actin mouse monoclonal antibody (Santa Cruz Biotechnology) as an internal control, and horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG as secondary antibodies, respectively.

Protease Inhibitor:

Article Title: GPC1 exosome and its regulatory mi RNAs are specific markers for the detection and target therapy of colorectal cancer
Article Snippet: The exosome pellets were resuspended in 1× PBS containing protease inhibitor cocktail (Roche). .. The membrane was blocked with 5% non‐fat dried milk for 1 hr and incubated overnight at 4°C with anti‐GPC1 antibody, anti‐CD63 antibody and anti‐β‐Actin Antibody (Santa Cruz), followed by incubation with horseradish peroxidase‐conjugated goat anti‐rabbit IgG (Cell Signaling, Danvers, MA, USA) secondary antibody for 2 hrs at RT.

Infection:

Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *
Article Snippet: Other antibodies used included horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen), horseradish peroxidase-conjugated anti-HA antibody (Roche Applied Science), polyclonal anti-actin antibody (clone C-11; Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Pierce). .. Viral Production and Infectivity Assay —HIV-1 virions were produced from 293T cells by standard calcium phosphate transfection.

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞
Article Snippet: Anti-JAK2 antibody, anti-Bcl-XL antibody, and anti-β-actin antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). .. Cell Cultures —BaF3 cells were infected with empty virus (MSCV), wild type JAK2 (WT) c-HA, or mutant JAK2 c-HA (L611S) with erythropoietin receptor c-FLAG and established as described previously ( ).

Recombinant:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞
Article Snippet: Reagents —Recombinant human Epo (ESPO®3000) was purchased from Kirin Brewery Co. (Tokyo, Japan). .. Anti-JAK2 antibody, anti-Bcl-XL antibody, and anti-β-actin antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

MTT Assay:

Article Title: Anti-cancer effects of Kochia scoparia fruit in human breast cancer cells
Article Snippet: Reagents and antibodies Paclitaxel, Hoechst 33342, MTT (3, 4, 5-dimethyl N-methylthiazol-2-yl-2, 5-d-phenyl tetrazolium bromide) and propidium iodide (PI) solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. The antibodies targeting cleaved caspase 3, cleaved caspase 8, cleaved caspase 9 and cleaved Poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA, USA), while anti-beta actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Fluorescence:

Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma
Article Snippet: Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β -actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA). .. Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β -actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA).

Mutagenesis:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞
Article Snippet: Anti-JAK2 antibody, anti-Bcl-XL antibody, and anti-β-actin antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). .. Cell Cultures —BaF3 cells were infected with empty virus (MSCV), wild type JAK2 (WT) c-HA, or mutant JAK2 c-HA (L611S) with erythropoietin receptor c-FLAG and established as described previously ( ).

Microscopy:

Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma
Article Snippet: Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β -actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA). .. Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β -actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA).

Mouse Assay:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞
Article Snippet: For administration to nude mice, AG490 was diluted in DMSO at a concentration of 25 mg/ml. .. Anti-JAK2 antibody, anti-Bcl-XL antibody, and anti-β-actin antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Blocking Assay:

Article Title: Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2 *
Article Snippet: The anti-β-actin antibody (sc-47778, 1:5,000) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). .. The appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, Santa Cruz Biotechnology, Inc.) were incubated in the blocking solution for 1 h at room temperature, followed by multiple washes with PBS-Tween 20.

SDS Page:

Article Title: Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition
Article Snippet: Proteins were separated by 10% SDS-PAGE and transferred to a methanol-activated PVDF membrane (GE Healthcare, Piscataway, NJ, USA). .. The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with anti-ATR antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-Chk1 antibody(Santa Cruz Biotechnologies), anti-pChk1 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-ATM antibody (Abcam, Cambridge, UK) and anti-β -actin antibody (Santa Cruz Biotechnologies) for 2 h. After 1-h incubation with goat-anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnologies), the protein bands were detected with luminal reagent (GE Healthcare) and their relative intensities were quantified using Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA).

Article Title: Notch1 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner
Article Snippet: Equal amounts of protein (150 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Roche). .. Antibodies were purchased from the following sources: anti-Notch1 antibody (Cell Signaling Technology, Boston, MA, USA), anti-NF-κB65 antibody (Cell Signaling Technology), anti-Hey1 antibody (Abcam, Cambridge, MA, USA), anti-Hes1 antibody (Cell Signaling Technology), anti-E-cadherin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Abcam), anti-vimentin antibody (Cell Signaling Technology), anti-occludin antibody (Proteintech Group Inc., USA), anti-Slug antibody (Abcam), anti-β-catenin antibody (Cell Signaling Technology), anti-Snail (Proteintech Group Inc.), anti-Twist antibody (Proteintech Group Inc.), anti-ZEB1 (Cell Signaling Technology), anti-ZEB2 (Abcam), anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LaminB1 antibody (Santa Cruz Biotechnology), anti-N2ICD (Santa Cruz Biotechnology), anti-N3ICD (Santa Cruz Biotechnology), and anti-N4ICD (Santa Cruz Biotechnology).

Plasmid Preparation:

Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *
Article Snippet: The Vif expression vector pNL-A1 and its control, pNL-A1ΔVif, were provided by K. Strebel (National Institute of Allergy and Infectious Diseases). .. Other antibodies used included horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen), horseradish peroxidase-conjugated anti-HA antibody (Roche Applied Science), polyclonal anti-actin antibody (clone C-11; Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Pierce).

Software:

Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma
Article Snippet: Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β -actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA). .. The fluorescence intensity of the negative control was subtracted and the level of Gremlin relative to β -actin was calculated as a percentage using image analysis software (Image Pro-Plus, ver.

Article Title: Repression of ATR pathway by miR-185 enhances radiation-induced apoptosis and proliferation inhibition
Article Snippet: .. The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with anti-ATR antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-Chk1 antibody(Santa Cruz Biotechnologies), anti-pChk1 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-ATM antibody (Abcam, Cambridge, UK) and anti-β -actin antibody (Santa Cruz Biotechnologies) for 2 h. After 1-h incubation with goat-anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnologies), the protein bands were detected with luminal reagent (GE Healthcare) and their relative intensities were quantified using Adobe Photoshop software (Adobe Systems Incorporated, San Jose, CA, USA). .. Flow cytometry assay Cells were fixed in −20 °C pre-chilled 70% ethanol overnight, and then stained with propidium iodide.

Article Title: Notch1 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner
Article Snippet: Signals were quantified using Image-Pro Plus 6.0 software (Media Cybernetics). .. Antibodies were purchased from the following sources: anti-Notch1 antibody (Cell Signaling Technology, Boston, MA, USA), anti-NF-κB65 antibody (Cell Signaling Technology), anti-Hey1 antibody (Abcam, Cambridge, MA, USA), anti-Hes1 antibody (Cell Signaling Technology), anti-E-cadherin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Abcam), anti-vimentin antibody (Cell Signaling Technology), anti-occludin antibody (Proteintech Group Inc., USA), anti-Slug antibody (Abcam), anti-β-catenin antibody (Cell Signaling Technology), anti-Snail (Proteintech Group Inc.), anti-Twist antibody (Proteintech Group Inc.), anti-ZEB1 (Cell Signaling Technology), anti-ZEB2 (Abcam), anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LaminB1 antibody (Santa Cruz Biotechnology), anti-N2ICD (Santa Cruz Biotechnology), anti-N3ICD (Santa Cruz Biotechnology), and anti-N4ICD (Santa Cruz Biotechnology).

Article Title: Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells
Article Snippet: The anti-β-Actin antibody (sc1615, Santa Cruz Biotechnology) or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GTX100118, Genetex) were used as a loading controls. .. Protein expression profiles were revealed with Clarity Western ECL Substrate (Biorad) and banding quantification was performed using the Quantity One Chemidoc XRS software (Biorad).

Article Title: The N-Ethyl-N-Nitrosourea-Induced Goldenticket Mouse Mutant Reveals an Essential Function of Sting in the In Vivo Interferon Response to Listeria monocytogenes and Cyclic Dinucleotides ▿
Article Snippet: Secondary anti-rabbit IRdye680 (LI-COR Biosciences, Lincoln, NE) and anti-mouse IRdye800 were used to detect primary antibodies, and the blots were analyzed using Oddessy analysis software (LI-COR). .. Overexpressed wild-type or Gt Sting was detected 24 h posttransfection using anti-hemagglutinin (anti-HA) antibody (Roche, Indianapolis, IN), and β-actin was detected using an anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Negative Control:

Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma
Article Snippet: .. Tissue microarrays were incubated with rabbit anti-human Gremlin polyclonal antibody (1 : 100 dilution), rabbit anti-β -actin monoclonal antibody (positive control; 1 : 100 dilution), or normal rabbit IgG (negative control; 0.1 lg/mL), followed by incubation with a secondary antibody (1 : 100 dilution; anti-rabbit IgG-FITC; all antibodies are from Santa Cruz Biotechnology, Santa Cruz, CA). .. Expression was examined by fluorescence microscopy (FMV 1000; Olympus, Tokyo, Japan).

Binding Assay:

Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells
Article Snippet: .. Nonspecific binding proteins were blocked with 3% skim milk in 1× phosphate-buffered saline with 0.05% Tween 20 for 60 min. Membranes were incubated with primary antibodies against antihuman IL-1β antibody (AF-201-NA, R & D Systems, Minneapolis, MN, USA), NF-κB sampler kit antibodies (#9936; Cell Signaling Technology; Danvers, MA, USA), and CEBP Antibody Sampler Kit (#12814, Cell Signaling Technology), PI3 Kinase Antibody Sampler Kit (#9655, Cell Signaling Technology), or anti-β-actin antibody (sc4778, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C. .. The membranes were probed further with horseradish peroxidase-conjugated secondary anti-sera (A9917, A6667, or A5420; Sigma-Aldrich Corp., St. Louis, MO, USA) and visualized with PierceFast Western blot kit (Thermo Fisher Scientific) and a cooled CCD camera system (Bio-Rad Laboratories, Hercules, CA, USA).

Quantitation Assay:

Article Title: Shotgun proteomics deciphered age/division of labor-related functional specification of three honeybee (Apis mellifera L.) exocrine glands
Article Snippet: Paragraph title: Immunoblotting and image quantitation ... Immunoblotting analyses were performed using anti-IDGF4 antiserum [ ], anti-human acetyl-CoA acyltransferase 2 rabbit polyclonal antibody (Sigma-Aldrich), anti-human aldolase rabbit polyclonal antibody (Cell Signaling Technology), and anti-50kDa protein (also known as MRJP2) antiserum [ ], and anti-beta-actin mouse monoclonal antibody (Santa Cruz Biotechnology) as an internal control, and horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG as secondary antibodies, respectively.

Produced:

Article Title: APOBEC3G Is Degraded by the Proteasomal Pathway in a Vif-dependent Manner without Being Polyubiquitylated *
Article Snippet: Other antibodies used included horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen), horseradish peroxidase-conjugated anti-HA antibody (Roche Applied Science), polyclonal anti-actin antibody (clone C-11; Santa Cruz Biotechnology), and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Pierce). .. Viral Production and Infectivity Assay —HIV-1 virions were produced from 293T cells by standard calcium phosphate transfection.

Concentration Assay:

Article Title: The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice *The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice * S⃞
Article Snippet: For administration to nude mice, AG490 was diluted in DMSO at a concentration of 25 mg/ml. .. Anti-JAK2 antibody, anti-Bcl-XL antibody, and anti-β-actin antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Lysis:

Article Title: Shotgun proteomics deciphered age/division of labor-related functional specification of three honeybee (Apis mellifera L.) exocrine glands
Article Snippet: Two lots of independent samples of PcGs, TGs and MGs, dissected from 10 nurse bees and 10 foragers, were homogenized and lysed in the aforementioned lysis solution, subjected to TCA precipitation, and then re-lysed in SDS-sample buffer (150 mM Tris-HCl, pH 6.8, containing 1.2% SDS and 30% glycerol). .. Immunoblotting analyses were performed using anti-IDGF4 antiserum [ ], anti-human acetyl-CoA acyltransferase 2 rabbit polyclonal antibody (Sigma-Aldrich), anti-human aldolase rabbit polyclonal antibody (Cell Signaling Technology), and anti-50kDa protein (also known as MRJP2) antiserum [ ], and anti-beta-actin mouse monoclonal antibody (Santa Cruz Biotechnology) as an internal control, and horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG as secondary antibodies, respectively.

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  • 98
    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: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin mab/product/Santa Cruz Biotechnology
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti β actin mab - by Bioz Stars, 2020-02
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    99
    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: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ß actin/product/Santa Cruz Biotechnology
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    99
    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: 99/100, based on 39 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
    Mouse Anti Human β Actin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-28799-8

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

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

    Techniques: Inhibition

    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