monoclonal anti beta actin antibody (Millipore)
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Monoclonal Anti beta Actin antibody
Description:
Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three α actins skeletal cardiac smooth one β actin β non muscle and two γ actins γ smooth muscle and γ non muscle ACTB actin β gene codes for β actin It is a cytoskeletal housekeeping protein It is present in the cytoplasm The ACTB gene is located on human chromosome 7p22 1 Monoclonal Anti β Actin mouse IgG2a isotype is derived from the AC 74 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse
Catalog Number:
a5316
Price:
None
Applications:
Western blot analysis of MDCK cell lysates were performed using monoclonal anti-actin antibody as a primary antibody.
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Structured Review
Millipore
monoclonal anti beta actin antibody
Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three α actins skeletal cardiac smooth one β actin β non muscle and two γ actins γ smooth muscle and γ non muscle ACTB actin β gene codes for β actin It is a cytoskeletal housekeeping protein It is present in the cytoplasm The ACTB gene is located on human chromosome 7p22 1 Monoclonal Anti β Actin mouse IgG2a isotype is derived from the AC 74 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse
https://www.bioz.com/result/monoclonal anti beta actin antibody/product/Millipore
Average 90 stars, based on 1592 article reviews
Price from $9.99 to $1999.99
Actin is a highly conserved protein that is a major component of both the cytoskeletal and contractile structures in all cell types It varies in amount being related to the type of differentiation and to the functional state of cells and tissues Actin can be found in two different forms of aggregation the globular or the fibrillar state and at least six distinct isoforms occur in vertebrates The actins exhibit over 90 sequence homology but each isoform has a unique NH2 terminal sequence The isoforms are comprised of three α actins skeletal cardiac smooth one β actin β non muscle and two γ actins γ smooth muscle and γ non muscle ACTB actin β gene codes for β actin It is a cytoskeletal housekeeping protein It is present in the cytoplasm The ACTB gene is located on human chromosome 7p22 1 Monoclonal Anti β Actin mouse IgG2a isotype is derived from the AC 74 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse
https://www.bioz.com/result/monoclonal anti beta actin antibody/product/Millipore
Average 90 stars, based on 1592 article reviews
Price from $9.99 to $1999.99
monoclonal anti beta actin antibody - by Bioz Stars,
2020-01
90/100 stars
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1) Product Images from "Extract of Ganoderma formosanum Mycelium as a Highly Potent Tyrosinase Inhibitor"
Article Title: Extract of Ganoderma formosanum Mycelium as a Highly Potent Tyrosinase Inhibitor
Journal: Scientific Reports
doi: 10.1038/srep32854
Figure Legend Snippet: GFE-EA inhibits tyrosinase activity and attenuates the protein level of tyrosinase in B16-F10 melanoma cells. ( a ) For assays of tyrosinase activity, 50 μg of total protein from B16-F10 melanoma cells was incubated with 2 mM L -DOPA, and the level of dopachrome was determined by a photometric method as described in Material and Methods and expressed as percentage of control. ( b ) Whole cell lysates, treated with 100–200 ppm of GFE-EA for 48 hours, were analyzed by western blotting with antibody against tyrosinase. Equal protein loading was confirmed by antibody against β-actin (full-length gels and blots are presented in Supplementary Figure S2 ).
Techniques Used: Activity Assay, Incubation, Western Blot
2) Product Images from "Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line, NCI-H28, with Homozygous Deletion of the β-Catenin Gene"
Article Title: Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line, NCI-H28, with Homozygous Deletion of the β-Catenin Gene
Journal: PLoS ONE
doi: 10.1371/journal.pone.0111550
Figure Legend Snippet: Neither β-catenin rescue nor deletion modify cell surface PAR 1 expression. NCI-H28 cells were transiently transfected with plasmide vector containing CTNNB1 or empty vector (Ctrl) while Met-5A cells were transfected with nonspecific (Ctrls) or specific β-catenin siRNA as described in Materials and Methods. A, relative expression levels of β-catenin. Transfected cells were lysed and total cell proteins were analysed by immunoblot using an anti-β-catenin antibody. Then membranes were reprobed with an anti-β-actin antibody. Data are expressed as arbitrary unit (fold variation over Ctrl) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences of β-catenin relative levels between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (*P≤0.05) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). B, a representative immunoblot. C, cell surface PAR 1 expression measured by ELISA assay. Antibody binding to fixed transfected cells was detected by horseradish peroxidise-conjugated secondary antibody. Data represent the mean ± SEM of three independent experiments performed in triplicate. The differences in cell surface PAR 1 expression between Ctrls and cell transfected with the recombinant vector or specific siRNA were significant (***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3).
Techniques Used: Expressing, Transfection, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay
Figure Legend Snippet: NCI-H28 cells over-express PAR 1 . A, relative expression levels of PAR 1 mRNA in Met-5A and NCI-H28 cells as determined by real time RT-PCR. B, relative expression levels of PAR 1 protein in primary mesothelial cells, Met-5A, NCI-H28, REN, Ist-Mes2, and Mero-14 cell lines as determined by immunoblot analysis followed by densitometric quantitation. Data are expressed as arbitrary unit (fold increase over Ctrl, Met-5A cells) after normalization by β-actin. Data shown are mean ± SEM of three independent experiments. The differences in PAR 1 expression levels between Ctrl (Met-5A or primary mesothelial cells) and MPM cells were significant (*P≤0.05, ***P≤0.001) by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3). C, a representative immunoblot.
Techniques Used: Expressing, Quantitative RT-PCR, Quantitation Assay
3) Product Images from "Genistein Disrupts Glucocorticoid Receptor Signaling in Human Uterine Endometrial Ishikawa Cells"
Article Title: Genistein Disrupts Glucocorticoid Receptor Signaling in Human Uterine Endometrial Ishikawa Cells
Journal: Environmental Health Perspectives
doi: 10.1289/ehp.1408437
Figure Legend Snippet: The GR and ER were required for transcriptional antagonism of three commonly regulated genes. ( A ) Cells treated 7 hr with the ER antagonist ICI or the GR antagonist RU486 assayed for ERα and GR protein levels by Western blot (left) and quantitated ER protein level (right). ( B ) mRNA expression of CA12 (carbonic anhydrase 12; top), LEFTY1 (left-right determination factor 1; center), and GILZ (glucocorticoid-induced leucine zipper; bottom) in cells pretreated 1 hr with ICI (left) or RU486 (RU; right) and then treated with vehicle (Veh), Dex, Gen, or Dex + Gen for 6 hr. ( C ) GR and ERα protein levels in cells transfected with non-targeting control (NTC) siRNA, GR siRNA, or ER α siRNA as assessed by Western blotting (left), and the extent of knockdown compared with NTC (right). ( D ) mRNA expression of CA12 (top), LEFTY1 (center), and GILZ (right) in cells transfected with NTC siRNA, GR siRNA, or ER α siRNA and treated for 6 hr with vehicle, Dex, Gen, or Dex + Gen. All protein values were normalized to β-actin and compared with vehicle-treated cells; all mRNA values were normalized to the housekeeping gene PPIB . Values are presented as mean ± SE of four biological replicates. * p
Techniques Used: Western Blot, Expressing, Transfection
4) Product Images from "Chk1 promotes replication fork progression by controlling replication initiation"
Article Title: Chk1 promotes replication fork progression by controlling replication initiation
Journal: Proceedings of the National Academy of Sciences of the United States of America
doi: 10.1073/pnas.1005031107
Figure Legend Snippet: Cdc7 codepletion rescues fork slowing in Chk1-depleted cells. ( A ) Protein levels of Cdc7, Chk1, and β-Actin (loading control) in U2OS cells after 48 hours depletion with Cdc7, Chk1, Cdc7, and Chk1 or control siRNA. ( B ) Quantification of origin firing in Cdc7-, Chk1- or control-depleted cells. First label origins (green-red-green) are shown as percentage of all red (CldU) labeled tracks. ( C ) Distribution of replication fork speeds in Cdc7- or control-depleted cells. ( D ) Distribution of replication fork speeds in Chk1- or control-depleted cells. ( E ) Distribution of replication fork speeds in Cdc7- or Cdc7 and Chk1-depleted cells. ( F ) Average replication fork speeds in Cdc7-, Chk1-, or control-depleted cells. Means and standard deviation (S.D.) (bars) of three independent experiments are shown. Values marked with asterisks are significantly different (student’s t -test, * p
Techniques Used: Labeling, Standard Deviation
5) Product Images from "Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus"
Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus
Journal: Environmental Health Perspectives
doi: 10.1289/EHP1575
Figure Legend Snippet: Effect of neonatal genistein exposure on ligand and GR expression. ( A ) Serum levels of corticosterone were measured in adult mice with intact ovaries and adrenal glands from serum collected between 0930 and 1130 hours in the morning. Data represent the mean of 11–12 animals ± SEM. ( B ) Relative mRNA expression of GR ( Nr3c1 ) was measured by qRT-PCR in adult ADX/OVX control and genistein-exposed mice. Expression was normalized to the reference gene Ppib and set relative to expression in control mice. GR protein levels, quantified by western blot analysis, were normalized to levels of the reference protein β-actin, which was not altered by treatment group (see Figure S5). GR protein levels were set relative to control mice. The results and image represent the mean of 4–5 animals ± SEM. ( C ) Representative images of GR expression and localization (red) in luminal epithelial and endometrial stromal cells. Hoescht 33342 was used to visualize nuclei (blue). Images were taken at 630 × . ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; GR, glucocorticoid receptor; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.
Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction
6) Product Images from "The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells"
Article Title: The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells
Journal: Journal of Experimental & Clinical Cancer Research : CR
doi: 10.1186/s13046-015-0206-x
Figure Legend Snippet: ZnCl 2 induces p53 nuclear accumulation and activation in the presence of low-dose ADR. ( a ) HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), for 4-8-16-24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53 and p21. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. One representative set of blot from three independent experiments, all generating similar results, is shown here. ( b ) RKO were treated with ZnCl 2 (100 μM), for 4-8-16-24 h and the expression levels of p53 was detected by western blotting. A positive control for p53 expression in the same cells, is included (ADR 2 μg/ml for 16 h). Anti-β-actin was used as protein loading control. ( c ) RKO and HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), 8 h. Equal amounts of nuclear extracts were separated by SDS-PAGE and p53 levels detected by western blotting. Anti-Lamin A was used as protein loading control. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here
Techniques Used: Activation Assay, Expressing, Western Blot, Positive Control, SDS Page
Figure Legend Snippet: ZnCl 2 increases the low-dose ADR-induced p53 stabilization in colon cancer cells. ( a ) RKO and ( b ) HCT116, plated under the same confluence condition, were treated with increasing doses (0.1 to 2 μg/ml) of ADR in the presence or absence of ZnCl 2 (100 μM), for 24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53, γH2AX, and PARP (cleaved form). The samples derive from the same experiment and gels/blots were processed in parallel. ( c ) The phosphorylation of p53 at Ser15 and Ser46 was detected in HCT116 treated with ADR (0.1-1-2 μg/ml) in the presence or absence of ZnCl 2 (100 μM) for 24 h, by western blotting. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here. ( d ) HCT116 cells were treated with ADR (0.2 μg/ml) and ZnCl 2 (100 μM) for 24 h. After treatments, total cell extracts were immunoprecipitated with anti-p53 antibody. Western blot analysis was performed with anti-p53 and anti-MDM2 antibodies. IP: immunoprecipitation. IB: immunoblotting
Techniques Used: Expressing, Western Blot, Immunoprecipitation
7) Product Images from "A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells"
Article Title: A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells
Journal: Journal of Experimental & Clinical Cancer Research : CR
doi: 10.1186/1756-9966-32-72
Figure Legend Snippet: Zn-curc restores wild-type p53-DNA binding and transactivating activities. (A) SKBR3 and U373 cells (6x10 6 ) were plated in 150 mm dish and the day after treated with Zn-curc (100 μM) for 16 h before assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1, MDM2) or for mtp53 target promoters (MDR1, cyclin B2). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). (B) Cells (3x10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24/48 h. p53 target genes were detected by RT-PCR analysis. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (C) SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc (100 μM) for 24 h, with or without p53 inhibitor pifithrin-α (PFT-α) (30 μM). p53 target genes were dtected by RT-PCR analysis. β-actin was used as control. (D) Gene expression as in (C) , was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for the indicated hours and total cell extracts were subjected to immunoblot analysis. (F) U373 cells were plated at subconfluence in 60 mm dish and the day after treated with curcumin (Curc) (50, 100 μM) for 24 h. Zn-curc (100 μM for 24 h) was used as control of p53 activation. p53 target genes were detected by RT-PCR. β-actin was used as control.
Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Activation Assay
Figure Legend Snippet: Zn-curc impairs survival of mutant p53-carrying cells. (A) Tumor cells (4 x 10 4 ) were plated in 60 mm dish and 24 h later treated with increased amount of Zn-curc (20, 50, 100 μM). Twenty-four hours later, plates were washed with PBS and fresh medium was added. Death-resistant colonies were stained with crystal violet 14 days later. (B) Death-resistant colonies as in (A) were counted and plotted as percentage ± SD of two independent experiments performed in duplicate. (C) Cells (3 x 10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24 and 48 h. Cell viability was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. (D) Cytofluorimetric analysis of the SubG1 peak evaluated by Propidium Iodide (PI) staining (upper panel) and microscopical analysis of SKBR3 cells, mock-treated or treated with Zn-curc (100 μM) for 24 h (lower panel). Percentage of apoptotic cells is shown ± SD of two independent experiments. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-PARP (cleaved form, 87 Kd) or anti-β-actin antibodies. (F) RKO cells were treated with Zn-curc (100 μM), ZnCl 2 (100 μM) or adryamicin (ADR, 2 μg/ml) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-γH2AX (phopho-Ser139) or anti-β-actin antibodies.
Techniques Used: Mutagenesis, Staining, Trypan Blue Exclusion Assay
Figure Legend Snippet: Zn-curc reactivates mtp53 in an orthotopic U373 glioblastoma model. (A) U373MG-LUC cells (2.5x10 5 ) were injected into the brain of athymic mice and left to growth for 6 days before treating animals with Zn-curc every day for 7 days. Mock- or Zn-curc-treated U373M-derived tumors were then harvested and analysed with a fluorescent microscope that showed as diffuse fluorescence only in Zn-curc - treated tumors. (B) Quantification of tumor cell fluorescence positivity in U373-derived tumors, untreated or Zn-curc-treated, ±SD. (C) Total mRNA was extracted from harvested U373-derived tumors, untreated or Zn-curc-treated, and p53 target gene expression as well as VEGF, MDR1 and Bcl2 expression were assayed by PCR of reverse-transcribed cDNA. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD.
Techniques Used: Injection, Mouse Assay, Derivative Assay, Microscopy, Fluorescence, Expressing, Polymerase Chain Reaction
8) Product Images from "Gentian violet induces wtp53 transactivation in cancer cells"
Article Title: Gentian violet induces wtp53 transactivation in cancer cells
Journal: International Journal of Oncology
doi: 10.3892/ijo.2014.2304
Figure Legend Snippet: Gentian violet (GV)-induced cell death is impaired by p53 inhibition. (A) ADF cells (2×10 5 ) were plated at subconfluence in 60-mm Petri dish and the day after treated with GV (0.2–1–2 μ M) for 24 h. Cell death was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. In the lower panel is shown western immunoblotting performed on equal amount of total cell extracts to detect PARP cleavage level. Anti-β-actin was used as protein loading control. (B) Semi-quantitative RT-PCR analyses of p53 expression in HCT116-p53 +/+ and -p53 −/− cells and in RKO-Ctr and -sip53 cells. β-actin was used as internal control. (C) Cell death analyses of the indicated cells were (2×10 5 ) at subconfluence in 60-mm Petri dish and the day after treated with GV (1 μ M) for 24 h. Cell death was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. * P
Techniques Used: Inhibition, Trypan Blue Exclusion Assay, Western Blot, Quantitative RT-PCR, Expressing
Figure Legend Snippet: Gentian violet (GV) induces DNA damage with p53 stabilization. (A) RKO and HCT116 cells were treated with GV (1 μ M) for 16 and 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect phospho-Histone H2A.X (γH2AX) levels. Adriamycin (ADR) (2 μ g/ml) was used as control of DNA damage. Anti-β-actin was shown as protein loading control. (B) RKO and HCT116 cells were treated with GV (1 μ M) 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect p53 levels. Anti-β-actin was used as protein loading control.
Techniques Used: Western Blot
Figure Legend Snippet: Gentian violet (GV) induces p53/DNA binding and transcriptional activity. (A) ADF, HCT116 and RKO cells (4×10 6 ) were plated in 150-mm dish and the day after treated with GV (1 μ M) for 16 h before assayed for chromatin immunoprecipitation (ChIP) analysis with anti-p53 (FL393) antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (IgG). (B) H1299 cells were co-transfected with Noxa-luc (1 μ g) reporter and wtp53 (0.5 μ g/sample) expression vector. Twenty-four hours after transfection GV (1 μ M) was added for 24 h before luciferase activity was assayed. The shown data represent the mean ± SD from three independent experiments performed in duplicate. Relative luciferase unit. (C, left panel) Semi-quantitative RT-PCR analyses of p53 target genes in H1299 cells transfected and treated with GV as in (B). β-actin was used as internal control. (C, right panel) Gene expression was measured by densitometric analyses and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ± SD. (D) HCT116 and ADF cells were plated at subconfluence in 60-mm Petri dish and the day after treated with GV (1 μ M) in the presence or absence of pifithryin-α (PFT-α) (30 μ M), for 24 h. P53 target gene expression was detected by RT-PCR analyses. β-actin was used as internal control. (E) RKO and ADF cells were treated with GV (1 and 2 μ M) for 24 h. Western immuno blotting was performed on equal amount of total cell extracts to detect Bax levels. Anti-β-actin was used as protein loading control.
Techniques Used: Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Amplification, Transfection, Expressing, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Western Blot
9) Product Images from "Neuroprotective Effects of Citicoline in in Vitro Models of Retinal Neurodegeneration"
Article Title: Neuroprotective Effects of Citicoline in in Vitro Models of Retinal Neurodegeneration
Journal: International Journal of Molecular Sciences
doi: 10.3390/ijms15046286
Figure Legend Snippet: Citicoline treatment does not induce cytotoxic effect in primary retinal cultures. Primary retinal cultures, treated or not with 100 μM citicoline for 96 h, were immunolabeled with antibodies against rhodopsin ( A , D rhod), GABA ( B , E ) and CRALBP ( C , F ), in order to evidence photoreceptors, GABAergic neurons and Müller glia, respectively. Cell nuclei were counterstained with Hoechst 33258. Antibody immunoreactivity was unaffected in citicoline-treated samples, showing that the agent does not influence cell composition and differentiation in primary retinal cultures ( D – F ); Apoptosis was evaluated by TUNEL assay. Apoptotic nuclei were quantified as percentage of TUNEL-positive nuclei over total nuclei. Bars represent the mean ± SEM of at least five independent experiments ( G ); Absence of proapoptotic effects after citicoline treatment is also shown by unchanged levels of activated cleaved caspase 3 ( H ). Whole cell lysates were prepared from primary retinal cultures. Equal amounts of total protein from each lysate were resolved on 15% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with anti-cleaved caspase 3. β-actin levels were used as control of protein loading. Blots are representative of three independent experiments. Bars = 20 μm.
Techniques Used: Immunolabeling, TUNEL Assay, SDS Page
10) Product Images from "Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7"
Article Title: Differentiation of Neurons Restricts Arbovirus Replication and Increases Expression of the Alpha Isoform of IRF-7
Journal: Journal of Virology
doi: 10.1128/JVI.02394-14
Figure Legend Snippet: SINV intracellular replication is delayed in differentiated AP-7 neurons. AP-7 neurons were infected with the TE strain of SINV (MOI, 5). (A) cAP-7 and dAP-7 neurons were fixed at 24 h after infection, and ultrastructure analysis was performed by transmission electron microscopy of thin sections. Bar, 100 nM. (B) Total cellular RNA was collected at various times after infection. cDNA was produced using SINV-specific primers and quantified by qPCR compared to standard SINV genomic DNA. SINV RNA levels in cAP-7 (dashed lines) or dAP-7 (solid lines) neurons are expressed as the mean SINV copy number (log 10 ) ± standard deviation of triplicate samples from a representative of two experiments. (C) Immunoblot analysis of total cell lysates prepared at the indicated times from mock-infected (M) or SINV-infected cAP-7 (left) or dAP-7 (right) neurons with anti-nsP3 (top), anti-E2 (middle), or anti-β-actin (bottom) antibodies. A representative of 2 experiments is shown. Significant differences between cAP-7 and dAP-7 at each time point were determined by two-way ANOVA with Bonferroni's posttest. ***, P
Techniques Used: Infection, Transmission Assay, Electron Microscopy, Produced, Real-time Polymerase Chain Reaction, Standard Deviation
Figure Legend Snippet: Irf-3 and Irf-7 mRNAs and IRF-7 protein increase during differentiation. (A) Total cellular RNA was collected at various times after plating of AP-7 neurons. Neurons were incubated at the permissive temperature in DMEM–10% FBS, 1 day prior to day 0 collection, corresponding to cAP-7 neurons. After day 0, neurons were maintained in differentiation medium at the nonpermissive temperature as described for the differentiation procedures. cDNA was produced using random primers, and Irf-3 and Irf-7 mRNAs were measured by qPCR and normalized to glyceraldehyde 3-phosphate dehydrogenase levels. mRNA levels are expressed as the mean value compared to levels in uninfected day 0 cAP-7 neurons ± standard deviations of six samples. (B) Total RNA was collected from day 0 (9 replicates) and day 7 (12 replicates) AP-7 neuron cultures or from triplicate cultures of day 0 or day 28 CSM14.1 neurons. mRNA levels, measured as described for panel A, are expressed as the mean value of differentiated cultures compared to levels in day 0 AP-7 or CSM14.1 neurons ± standard deviations. (C) Total cell lysates prepared from AP-7 neurons during differentiation were analyzed by immunoblot analysis with anti-IRF-7 (top row), anti-IRF-3 (middle row), or anti-β-actin (bottom row) antibodies. Multiple isoforms of IRF-7 are detected by the IRF-7 antibody. (D) Total cell lysates prepared from CSM neurons at the indicated days during differentiation were analyzed by immunoblot analysis with anti-IRF-7 (top row) or anti-β-actin (bottom row) antibodies. (E) Brain homogenates (10%) from 3-day-old or 6-week-old C57BL/6J mice were analyzed by immunoblot analysis with anti-IRF-7 (top row), anti-IRF-3 (middle row), or anti-β-actin (bottom row) antibodies. Results of a representative experiment of at least 2 experiments are shown. Significant differences between cAP-7 and dAP-7 at each time point were determined by one-way ANOVA with Dunnett's posttest. ***, P
Techniques Used: Incubation, Produced, Real-time Polymerase Chain Reaction, Mouse Assay
Figure Legend Snippet: Stat1 is not phosphorylated during SINV infection of AP-7 neurons. (A) cAP-7 or dAP-7 neurons were mock infected (M) or infected with SINV (MOI, 5). Total cell lysates prepared at the indicated times after infection were analyzed with anti-pSTAT1 (P-Y701) (top row), anti-STAT1 (middle row), or anti-β-actin (bottom row) antibodies. Results of a representative experiment of 2 experiments are shown. (B) cAP-7 or dAP-7 neurons were treated with 500 IU/ml of recombinant rIFN-α, -β, or -γ. Total cell lysates prepared 4 h after treatment were analyzed by immunoblotting using anti-STAT1 (P-Y701) (top row) or anti-β-actin (bottom row) antibodies. Results of a representative experiment of 2 experiments are shown.
Techniques Used: Infection, Recombinant
11) Product Images from "Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival"
Article Title: Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival
Journal: PLoS ONE
doi: 10.1371/journal.pone.0118311
Figure Legend Snippet: Deletion of SNAP-23 leads to acute death of MEFs. MEF lines were generated from SNAP-23 fl/+ mice (clones 75.1 and 88.2) or SNAP-23 fl/- mice (clones 75.2 and 88.3). (A) Infection of a typical SNAP-23 fl/+ MEF culture infected with empty retrovirus (left panel) or GFP-Cre retrovirus (right panel) shows that two days after infection the vast majority of MEFs were viable (Annexin V - ) and expressed GFP-Cre. (B) The indicated MEF lines were infected with GFP-Cre-expressing retrovirus and the number of live cells present in each culture (based on staining with PI) was determined at different times. The absolute cell recovery in each condition was expressed relative to the amount of cells present two days after infection (control experiments showed that there was no Cre-dependent cell death in any line after only two days of infection). The data shown are representative of two independent experiments analyzed at day 2, 4, 6, 8 and one experiment analyzed at day 1, 3, 5, 7. (C) Adherent SNAP-23 fl/- MEFs were isolated 4 days after retroviral transduction with Cre (Cre+) or after mock-transduction (Cre-). Equal numbers of cells from each culture were analyzed by SDS-PAGE and immunoblotting using a SNAP-23 antibody. The blot was re-probed for anti-β actin mAb as a loading control. The amount of SNAP-23 present in each cell lysate was normalized to the amount of actin present and the data shown are mean +/- SD of three independent experiments (*p
Techniques Used: Generated, Mouse Assay, Infection, Expressing, Staining, Isolation, Transduction, SDS Page
12) Product Images from "Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats"
Article Title: Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats
Journal: Molecular Pain
doi: 10.1177/1744806918803039
Figure Legend Snippet: Alterations of P2X 7 receptor protein level in the vlPAG induced by cancer cell inoculation or tramadol injections. (a) By using Western blot analysis, we detected a protein band of ≈68 kDa, coinciding with the known molecular weight of the P2X 7 receptor. (b) β-actin was used as loading control. The P2X 7 receptor protein levels in different groups were expressed as ROD. No distinct change in P2X 7 receptor protein level was observed in vlPAG between normal and sham-BCP groups. The P2X 7 receptor protein level in vlPAG in BCP group was increased compared with normal and sham-BCP groups. Intraperitoneal tramadol injection at doses of 10, 20, and 40 mg/kg showed significantly and dose dependently upregulated the P2X 7 receptor protein level in vlPAG. n = 6; ⋆⋆ p
Techniques Used: Western Blot, Molecular Weight, Injection
13) Product Images from "Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats"
Article Title: Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats
Journal: Molecular Pain
doi: 10.1177/1744806918803039
Figure Legend Snippet: Alterations of P2X 7 receptor protein level in the vlPAG induced by cancer cell inoculation or tramadol injections. (a) By using Western blot analysis, we detected a protein band of ≈68 kDa, coinciding with the known molecular weight of the P2X 7 receptor. (b) β-actin was used as loading control. The P2X 7 receptor protein levels in different groups were expressed as ROD. No distinct change in P2X 7 receptor protein level was observed in vlPAG between normal and sham-BCP groups. The P2X 7 receptor protein level in vlPAG in BCP group was increased compared with normal and sham-BCP groups. Intraperitoneal tramadol injection at doses of 10, 20, and 40 mg/kg showed significantly and dose dependently upregulated the P2X 7 receptor protein level in vlPAG. n = 6; ⋆⋆ p
Techniques Used: Western Blot, Molecular Weight, Injection
14) Product Images from "Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus"
Article Title: Neonatal Genistein Exposure and Glucocorticoid Signaling in the Adult Mouse Uterus
Journal: Environmental Health Perspectives
doi: 10.1289/EHP1575
Figure Legend Snippet: Effect of neonatal genistein exposure on ligand and GR expression. ( A ) Serum levels of corticosterone were measured in adult mice with intact ovaries and adrenal glands from serum collected between 0930 and 1130 hours in the morning. Data represent the mean of 11 – 12 animals ± SEM . ( B ) Relative mRNA expression of GR ( Nr3c1 ) was measured by qRT-PCR in adult ADX/OVX control and genistein-exposed mice. Expression was normalized to the reference gene Ppib and set relative to expression in control mice. GR protein levels, quantified by western blot analysis, were normalized to levels of the reference protein β -actin , which was not altered by treatment group (see Figure S5). GR protein levels were set relative to control mice. The results and image represent the mean of 4 – 5 animals ± SEM . ( C ) Representative images of GR expression and localization (red) in luminal epithelial and endometrial stromal cells. Hoescht 33342 was used to visualize nuclei (blue). Images were taken at 630 × . ADX/OVX, adrenalectomized/ovariectomized; Dex, dexamethasone; GR, glucocorticoid receptor; qRT-PCR, quantitative real-time polymerase chain reaction; Veh, vehicle.
Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction
15) Product Images from "Anticancer activity of cationic porphyrins in melanoma tumour-bearing mice and mechanistic in vitro studies"
Article Title: Anticancer activity of cationic porphyrins in melanoma tumour-bearing mice and mechanistic in vitro studies
Journal: Molecular Cancer
doi: 10.1186/1476-4598-13-75
Figure Legend Snippet: Effect of porphyrins on protein RAS and ERK pathway . (A) Immunoblots showing the expression level of protein RAS in melanoma B78-H1 cells 24 h after treatment with porphyrins P2, P4 and C14 (1 and 5 μM) without irradiation. C14 at 5 μM reduces the level of protein RAS through a light-independent mechanism; (B) Immunoblots showing the levels of RAS, ERK, P-ERK, NF-κB and β-actin in untreated or porphyrin/light treated B78-H1 cells, 24 h after light treatment.
Techniques Used: Western Blot, Expressing, Irradiation
Figure Legend Snippet: Annexin V-propidium iodide and PARP-1 cleavage assays . (A) Annexin V-propidium iodide assay of B78-H1 cells treated with 250 nM P2 or P4 and 10 nM C14. The proportion of cell population in apoptosis and necrosis is reported in Table 1 . Before the FACS analysis the cells have been treated with porphyrin and light (7.2 J/cm 2 ). The experiment has been performed in triplicate; (B) Levels of PARP-1 and β-actin in B78-H1 cells untreated or treated with 80 and 150 nM P2 and P4, 7.5 nM C14. The Western blots show the cleavage of PARP-1.
Techniques Used: FACS, Western Blot
16) Product Images from "The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer *The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer * ♦"
Article Title: The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer *The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer * ♦
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.M115.661553
Figure Legend Snippet: BAP1 interacts with ASXL1/2 via its CTD domain. A, schematic representation of the BAP1 fragments used for in vitro pulldown in B. B, GST-pulldown assay using GST-BAP1 fragments and methionine 35 S-labeled ASXM domains of ASXL1 or ASXL2. The arrows indicate the full-length forms of the fragments. C, schema of the different deletions in the CTD domain used to generate BAP1 mutants. BAP1 ΔCTD1 ). We also generated a BAP1 ΔCTD , which represents a mutant with a deletion of the CTD domain (Δ631–693 amino acids). BAP1 ΔCC2 represents a mutant with a smaller deletion within the CTD domain (Δ635–655 amino acids). D, functional CTD is required for proper protein stability of BAP1. Protein expression levels of BAP1 and its CTD deletion mutant form in stable HeLa S cell lines are shown ( top panel ). Myc-BAP1, Myc-BAP1 ΔCTD expression constructs (3 μg each) were transfected in 293T cells, which were harvested, 3 days post-transfection, for immunoblotting ( bottom panel ). E, left panel , silver stain of the immunopurified BAP1 and BAP1 ΔCTD1 complexes. Right panel , Western blot detection of components of the BAP1 complexes. The high and low arrows indicate the position of ASXL2 and BAP1 (WT and BAP1 ΔCTD1 ), respectively. β-Actin or YY1 are used as protein loading controls. The dot indicates a monoubiquitinated form of BAP1 ( D ).
Techniques Used: In Vitro, GST Pulldown Assay, Labeling, Generated, Mutagenesis, Functional Assay, Expressing, Construct, Transfection, Silver Staining, Western Blot
Figure Legend Snippet: BAP1 and ASXL1/2 are co-regulated, and loss of BAP1 in cancer is concomitant with ASXL2 depletion. A, 293T cells were transfected with BAP1 and increasing amounts of either Myc-ASXL1 (0.5, 1, 2, and 5 μg) or Myc-ASXL2 (0.5, 1, 2, and 5 μg) expression vectors and harvested, 3 days later, for immunoblotting. B, 293T cells were transfected with Myc-ASXL1 or Myc-ASXL2 with increasing amounts of BAP1 (0.3 and 1 μg) vectors and harvested, 3 days later, for immunoblotting. Quantification of band intensity for each protein was conducted relative to the lowest amount of transfected plasmid ( A and B ). C, protein levels following siRNA depletion of BAP1 and/or ASXL1/2 in U2OS cells. D, protein expression following siRNA depletion of BAP1, ASXL1, and ASXL2 in LF1 human fibroblasts. E, depletion of endogenous BAP1 using siRNA in U2OS cells stably expressing empty vector, siRNA-resistant BAP1 wild type, or siRNA-resistant BAP1 catalytic dead mutant (C91S). Protein levels of BAP1 and ASXL2 were detected by immunoblotting. Quantification of band intensity was conducted relative to the non-target siRNA control ( C–E ). F, immunodepletion of BAP1 or ASXL2 from HeLa nuclear extracts. The nuclear DNA damage signaling enzyme, PARP1, was used as a control, which mostly remained in the flow-through fraction. G, reconstitution by retroviral infection of H28 mesothelioma and H226 non-small lung carcinoma BAP1-deficient cells with BAP1 or BAP1 C91S . Protein levels of BAP1 and mutants were detected by immunoblotting. H, mRNA of ASXL2 in reconstituted H226 cells was quantified by quantitative PCR. The data represent two independent experiments. β-Actin or YY1 are used as protein loading controls. Quantification of band intensity was conducted relative to BAP1-transfected samples ( G ). The dot and asterisks ) and nonspecific bands, respectively ( A–E and G ).
Techniques Used: Transfection, Expressing, Plasmid Preparation, Stable Transfection, Mutagenesis, Flow Cytometry, Infection, Real-time Polymerase Chain Reaction
Figure Legend Snippet: BAP1 regulates cell cycle progression in CTD-dependent manner. A, protein levels following depletion of endogenous BAP1 using siRNA in U2OS cells stably expressing siRNA-resistant BAP1, BAP1 C91S , or BAP1 R666-H669 . B and C, mutations of CTD disrupts BAP1 function in regulating cell proliferation. Following siRNA for endogenous BAP1, U2OS cells stably expressing siRNA-resistant BAP1, BAP1 C91S , BAP1 R666-H669 or BAP1 ΔCTD were synchronized by double thymidine block at the G 1 /S boundary and released 7 h to progress through S phase and were then subjected to FACS analysis. D, H226 BAP1-null cells stably expressing BAP1, BAP1 C91S , or BAP1 R666-H669 was analyzed by immunoblotting ( top panel ). Similar numbers of cells were plated and cultured for 5 days prior staining with crystal violet dye ( bottom panel ). YY1 and β-actin were used as a protein loading controls. The dot indicates a monoubiquitinated form of BAP1 ( A and D ).
Techniques Used: Stable Transfection, Expressing, Blocking Assay, FACS, Cell Culture, Staining
17) Product Images from "Puerarin attenuates cisplatin-induced rat nephrotoxicity: The involvement of TLR4/NF-κB signaling pathway"
Article Title: Puerarin attenuates cisplatin-induced rat nephrotoxicity: The involvement of TLR4/NF-κB signaling pathway
Journal: PLoS ONE
doi: 10.1371/journal.pone.0171612
Figure Legend Snippet: Expression of TLR4 and NF-κB p65 proteins in kidney tissues in each groups. (A) Representative Western blot picture, showing the expression levels of TLR4 and NF-κB p65 proteins. β-actin was used as an internal control. (B) Changes in the expression level of TLR4 protein. (C) Changes in the expression level of NF-κB p65 protein. Data are presented as mean ± SD (n = 4). ***P
Techniques Used: Expressing, Western Blot
18) Product Images from "Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABAA receptor δ subunit in cerebellar granule neurons and delays motor development in rats"
Article Title: Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABAA receptor δ subunit in cerebellar granule neurons and delays motor development in rats
Journal: Neuropharmacology
doi: 10.1016/j.neuropharm.2013.11.020
Figure Legend Snippet: Validation of anti-GABA A R δ subunit antibody Confocal microscopy images of 16 μm cerebellar vermis parasagittal sections from wildtype C57BL/6 (WT) and δ-subunit knockout (δ-KO) adult mice magnified at 20× (A and B) and 63× (C and D) . Each panel shows the GABA A R δ subunit (red), Pan Neuronal marker (green), nuclear counterstaining diamidino-2-phenylindole (DAPI; blue), and merge of the three markers. Cerebellar layers are indicated for each merged picture (molecular layer = ML; Purkinje cell layer = PCL; granule cell layer = GCL). Scale bar = 10 μm. Images from WT show intense staining for δ subunit, specifically in the GCL. δ-KO mice show no staining for δ subunit in the GCL. Same results obtained with sections from another pair of WT and δ-KO mice (not shown). (E) Results of western blot studies with anti- GABA A R δ subunit antibodies, which recognized closely spaced bands at ~53 kDa in homogenates from two different WT mice but not δ-KO adult mice. A non-specific band of ~80 kDa was recognized in tissue from WT and δ-KO mice. The lower panel shows β-actin expression levels. Each lane corresponds to a sample from a different mouse.
Techniques Used: Confocal Microscopy, Knock-Out, Mouse Assay, Marker, Staining, Western Blot, Expressing
Figure Legend Snippet: Characterization of GABA A -δ subunit expression by western immunoblotting (A) Exemplar western blots illustrating that GABA A R δ subunit levels increase with age in samples from air controls (CON) and postnatal alcohol exposed (PAE) rats (upper panel). Conversely, β-actin levels decreased with age (middle panel). An average of randomly selected Coomassie-stained bands was used to control for differences in protein loading (lower panel). (B) Summary of results ( n = 5–7 litters). PAE significantly increased GABA A R δ subunit levels at P28 (* - p
Techniques Used: Expressing, Western Blot, Staining
19) Product Images from "Expression of β-transducin repeat-containing E3 ubiquitin protein ligase in human glioma and its correlation with prognosis"
Article Title: Expression of β-transducin repeat-containing E3 ubiquitin protein ligase in human glioma and its correlation with prognosis
Journal: Oncology Letters
doi: 10.3892/ol.2015.3113
Figure Legend Snippet: Expression of β-TrCP in glioma tissues and non-tumorous human brain tissues. (A) Representative western blot analysis of the total extracts isolated from human brain glioma tissues (eight samples shown) and non-tumorous tissues (four samples shown) using human β-TrCP antibody. β-actin served as the internal control. (B) Quantitative analysis of the protein levels of β-TrCP in glioma tissues (n=32) and non-tumorous brain tissues (n=16), represented as a bar graph. *P
Techniques Used: Expressing, Western Blot, Isolation
20) Product Images from "Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting ☆"
Article Title: Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting ☆
Journal: Neural Regeneration Research
doi: 10.3969/j.issn.1673-5374.2012.13.004
Figure Legend Snippet: Tumor spheres expressed a high level of tumor suppressor phosphatase and tensin homolog (PTEN). Protein extracted from tumor spheres and attached differentiated cells, was analyzed by western blot using mouse anti-PTEN antibody or mouse anti-β-actin antibody.
Techniques Used: Western Blot
21) Product Images from "Silencing of CXCR4 Blocks Breast Cancer Metastasis"
Article Title: Silencing of CXCR4 Blocks Breast Cancer Metastasis
Journal: Cancer research
doi:
Figure Legend Snippet: CXCR4 expression levels of MDA-MB-231 cells at 48 hours posttransfection of CXCR4 siRNA-transfected MDA-MB-231 cells were stained with immunofluorescence by using biotinylated CXCR4 antagonistic peptide and streptavidin-phycoerythrin. Red, CXCR4 phycoerythrin staining; blue, counterstaining of nuclei. B, RT-PCR analysis of CXCR4 of the siRNA-transfected MDA-MB-231 cells showed that siRNA1+2 effectively blocked the expression of CXCR4 mRNA. Western blot results of the siRNA-transfected MDA-MB-231 cells by using anti-CXCR4 antibody Ab-2 (1:1,000) showed that siRNA1+2 blocked the expression of CXCR4 protein almost completely. β-Actin (Sigma, 1:2,500) was used as a loading control. Cont, control.
Techniques Used: Expressing, Multiple Displacement Amplification, Transfection, Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot
22) Product Images from "Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation"
Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na+, K+/ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation
Journal: Journal of Virology
doi: 10.1128/JVI.01861-17
Figure Legend Snippet: Digitoxin induces autophagic and proteasomal degradation of the α1 subunit. (A) HFFs were infected and treated with digitoxin (30 nM), AICAR (0.8 mM), digitoxin plus AICAR, or GCV (5 μM). Cells were treated with MG132 (10 μM) 10 h before harvest. Lysates were used to detect p-AMPK and α1. Na/K α1 subunit expression was normalized against β-actin expression. The ratios of normalized α1 levels in MG132-treated and untreated samples were quantitated by densitometry. (B) Infected and treated HFFs were immunoprecipitated with anti-ubiquitin antibody and probed for the Na + ,K + /ATPase α1 subunit in the presence of MG132 (10 μM). (C) HCMV-infected cells were treated or not treated with digitoxin. At 24 hpi, cycloheximide (CHX; 100 μg/ml) was added. Cells were harvested at the indicated time intervals following CHX addition for SDS-PAGE analysis to measure α1 subunit degradation. (D) Infected and digitoxin-treated cells were treated with bafilomycin A1 (50 nM) for 4 h before harvest. Lysates were prepared at 48 hpi, and levels of the Na + ,K + /ATPase α1 subunit and p62 were detected by WB. (E) Model depicting the mechanism of digitoxin-mediated autophagy induction and HCMV inhibition. (i and ii) HCMV activates mTOR (i), which suppresses ULK1 by phosphorylating it at Ser757 to inhibit autophagy (ii), creating an intracellular microenvironment favorable for HCMV replication. (iii) Digitoxin degrades the α1 subunit by ubiquitination and autophagy. (iv) AMPK activation through the α1 subunit is independent of α1 degradation. (v) pAMPK phosphorylates ULK1 at Ser317 and interacts with it to induce autophagy, which is detrimental for HCMV.
Techniques Used: Infection, Expressing, Immunoprecipitation, SDS Page, Western Blot, Inhibition, Activation Assay
Figure Legend Snippet: Digitoxin-mediated inhibition of mTOR activity in noninfected ATG5 KD cells is lost in HCMV-infected ATG5 KD cells. Infected (A and B) or uninfected (C) control and ATG5 KD cells were treated with digitoxin (30 nM) or GCV (5 μM) for the indicated time points, and cell lysates were used to detect viral proteins IE1/2 and UL44 and cellular proteins ATG5, p-mTOR, mTOR, p-Ser S6K, p-Thr S6K, S6K, and p4E-BP-1. β-Actin was used as a loading control.
Techniques Used: Inhibition, Activity Assay, Infection
Figure Legend Snippet: Digitoxin induces AMPK phosphorylation and autophagy flux in infected and noninfected HFFs. (A) Cells were infected with HCMV (MOI of 1 PFU/cell) and treated with digitoxin (30 nM) or GCV (5 μM) (I) or noninfected and treated with digitoxin or GCV (II). Lysates were prepared at the indicated time points and used for detection of pAMPK, AMPK, and IE1/2 by WB. (B) Model showing the function of bafilomycin A1 in inhibiting autophagosome-lysosome fusion, resulting in accumulation of LC3-II and p62 instead of their degradation. (C) Cells were treated as described for panel A for the indicated times. Bafilomycin A1 (50 nM) was added 4 h before harvest, and the levels of LC3-II and p62 were detected by WB. β-Actin was used as a loading control. (D) Cells were infected or treated with digitoxin (30 nM). LC3 puncta were detected by confocal microscopy at 24 and 72 hpi. The total number of LC3-II puncta/field was estimated by use of ImageJ software. The mean ± SD for three fields is provided for each condition. (E) HFFs were treated with digitoxin or GCV. Cell lysates were collected at the indicated time points for detection of pAMPK and p62 by WB. β-Actin was used as a loading control. Blots are the best representatives of three independent experiments.
Techniques Used: Infection, Western Blot, Confocal Microscopy, Software
Figure Legend Snippet: AICAR antagonizes HCMV inhibition by digitoxin in conjunction with reduced AMPK phosphorylation and autophagy. (A) HFFs were infected with HCMV (100 PFU/well), followed by treatment with compounds at the indicated doses. (Left) Digitoxin plus AICAR; (center) digitoxin plus CC; (right) digitoxin plus GCV. The effects of drug combinations were analyzed by WB at 24 hpi (B) and 72 hpi (C) for LC3-II, pAMPK, AMPK, UL44, and pp65. (D) Control and ATG5 KD cells were infected with HCMV (MOI = 1) and treated with AICAR (0.8 mM), digitoxin (30 nM), or GCV (5 μM). Lysates were prepared to detect p62 and viral pp65. β-Actin was used as a loading control. Blots are representative of three independent experiments.
Techniques Used: Inhibition, Infection, Western Blot
23) Product Images from "Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells"
Article Title: Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkr1242
Figure Legend Snippet: Cdx2 does alter neither Ku proteins and DNA-PKcs recruitment nor DNA-PK autophosphorylation but inhibits DNA-PKcs activity. ( A ) Cdx2 does not interfere with DSB Ku70 binding. HCT116 cells were transfected with pFlag (light gray) or Cdx2 (dark gray) expressing plasmid and Ku70 binding activity was assessed. Results represent the mean of at least four independent experiments. pFlag values were set at 1. ( B ) Cdx2 does not alter DNA-PKcs recruitment. HCT116 were transfected with either pFlag or pFlag-Cdx2 as indicated and immunoprecipitation was performed using anti-Ku70 antibodies in the presence or absence of Ethidium Bromide (EtBr) as indicated at the bottom of the figure. DNA-PKcs, Ku70 or Cdx2 were revealed by western blot. Twenty micrograms of whole-protein extract were loaded on the right line (input). ( C ) Cdx2 does not modify DNA-PKcs autophosphorylation. Time course analysis of the phosphorylation of the DNA-PKcs after etoposide treatment 100 μM for 1 hour of HCT116 cells transfected with pFlag or pFlag-Cdx2. Phospho-DNA-PKcs was revealed by western blot. β-actin was used to normalized the amount of loaded proteins. ( D ) Cdx2 inhibits DNA-PKcs activity. DNA-PKcs activity was assessed in HT29-TW6 or -TG8 (control) cells treated or not with etoposide (VP16) 100 μM or neocarzinostatin 200 ng/μL for 1 hour. Cdx2 expression was induced with doxycyclin when indicated (dark gray bars). Experiments without doxycyclin and etoposide treatment were considered as references and results were set at 1. Results illustrate the mean of at least six experiments and error bars represent SEM. Asterisk indicates a significant difference (** P
Techniques Used: Activity Assay, Binding Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot
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After incubation with the anti-BDNF primary antibody (sc-546, Santa Cruz, USA), Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients Article Snippet: Equal loading and transfer were assessed by reprobing the blots with Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or Expressing:Article Title: Effects of Balance and Gait Training on the Recovery of the Motor Function in an Animal Model of Parkinson's Disease Article Snippet: To confirm if the equivalent protein, Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients Article Snippet: Equal loading and transfer were assessed by reprobing the blots with BIA-KA:Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study Article Snippet: Sample lysates were centrifuged at 15,000 rpm for 20 min at 4 °C and protein concentration in the supernatants was determined by the bicinchoninic acid (BCA; Pierce, Rockford, IL, USA) microplate method. .. The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti Western Blot:Article Title: Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells Article Snippet: Paragraph title: Western blot analysis ... Antibodies against H3 (ab1791), DNMT3A (ab2850), and SUV39h1 (ab12405) were purchased from Abcam Inc. (Cambridge, UK); DNMT1 (sc-20701) and DNMT3B (sc-10235) from Santa Cruz Biotech (Santa Cruz, CA); Myc epitope tag (05-724) from Upstate (now Millipore, Billerica, MA) and G9a (G 6919); Article Title: Transcriptomic analysis reveals sex-specific differences in the expression of Dcl1 and Fis1 genes in the radio-adaptive response of thymocytes to TRP53-mediated apoptosis Article Snippet: Paragraph title: Western blot analysis ... For Article Title: LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence Article Snippet: .. Protein was isolated from cultured cortical neurons and western blot analyses performed as previously described, with antibodies directed toward cleaved caspase-3 (Cell Signaling, Cat No. 9664S), Rb (PharMingen, Cat No. 554136), Cyclin A2 (Abcam, Cat No. ab7965), PCNA (Millpore, Cat No. MAB424) and Article Title: High fat diet increases melanoma cell growth in the bone marrow by inducing osteopontin and interleukin 6 Article Snippet: Paragraph title: Western blot analysis ... Following protein transfer into 0.45 μM nitrocellulose (NC) membrane (BD, Cat.#1620115), membranes were blocked with 5% skim-milk in TBS-T buffer (0.05%) for 2 h. Then, membranes that washed and were probed using the following antibodies: Nuclear factor kappa B (NF-κB) p65 (Cell signaling Technology, Inc. Cat.#8242; 1:500 dilutions), phospho-NF-κB p65 (Ser468) (Cell signaling Technology, Inc. Cat.#3039; 1:500 dilutions) and Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke Article Snippet: The hippocampus was homogenized in lysis buffer B (137 mM NaCl, 20 mM Tris, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 0.5 mM sodium vanadate, pH 8.0) for western blot analysis. .. After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study Article Snippet: Paragraph title: 4.10. Western Blotting ... The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti Article Title: The Effects of Antecedent Exercise on Motor Function Recovery and Brain-derived Neurotrophic Factor Expression after Focal Cerebral Ischemia in Rats Article Snippet: The hippocampus was homogenized in lysis buffer B (137 mM NaCl, 20 mM Tris, 1% NP40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/Ml aprotinin, 1 µg/Ml leupeptin, 0.5 mM sodium vanadate, pH8.0) for western blot analysis. .. After incubation with the anti-BDNF primary antibody (sc-546, Santa Cruz, USA), Article Title: Escherichia coli Subtilase Cytotoxin Induces Apoptosis Regulated by Host Bcl-2 Family Proteins Bax/Bak ▿ Article Snippet: Paragraph title: Western immunoblotting. ... Anti-Puma (catalog no. P-4618) and Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients Article Snippet: Paragraph title: Western blot analysis ... Equal loading and transfer were assessed by reprobing the blots with Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: Paragraph title: Western blot analysis ... A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or Electrotransfer:Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: After the electrotransfer of the proteins, CRMP2 protein was detected using a rabbit antibody directed against CRMP2 (1:500; Abcam, Cat#ab36201, RRID:AB_731750) and the different synaptic markers were detected using mouse monoclonal antibodies directed against PSD-95 (1:20,000; Millipore, Merck, Cat#P246, RRID:AB_260911), synaptophysin (1:2000–1:20,000; Sigma, Cat#S5768, RRID:AB_477523), and syntaxin 1 A (1:2000; Sigma, Cat#S0664, RRID:AB_477483), guinea-pig polyclonal anti-vGluT1 (1:10,000; Synaptic Systems, Goettingen, Germany, Cat#135304 RRID:AB_887878), anti-vGAT (1:1000; Synaptic Systems; Cat#131004, RRID:AB_887873), and rabbit polyclonal anti-gephyrin (1:10,000; Abcam, Cat#ab25784 RRID:AB_1209349), all diluted in TBS-T (Tris-buffered saline, 0.1% Tween 20) containing 5% dried milk. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or TCA Precipitation:Article Title: Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells Article Snippet: Western blot analysis Proteins from the same volume of each fraction (200 to 250 μl) were concentrated by TCA precipitation, dissolved in SDS/β-mercaptoethanol loading buffer, and resolved on a 4% to 15% gradient SDS/PAGE gel (Bio-Rad, Hercules, CA). .. Antibodies against H3 (ab1791), DNMT3A (ab2850), and SUV39h1 (ab12405) were purchased from Abcam Inc. (Cambridge, UK); DNMT1 (sc-20701) and DNMT3B (sc-10235) from Santa Cruz Biotech (Santa Cruz, CA); Myc epitope tag (05-724) from Upstate (now Millipore, Billerica, MA) and G9a (G 6919); Cell Culture:Article Title: LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence Article Snippet: .. Protein was isolated from cultured cortical neurons and western blot analyses performed as previously described, with antibodies directed toward cleaved caspase-3 (Cell Signaling, Cat No. 9664S), Rb (PharMingen, Cat No. 554136), Cyclin A2 (Abcam, Cat No. ab7965), PCNA (Millpore, Cat No. MAB424) and Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: Protein extracts from cultured hippocampal neurons or hippocampal homogenates were diluted in 6× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer ((0.5 M Tris, 0.4% SDS, pH 6.8), 30% glycerol, 10% SDS, 0.6 M dithiothreitol, and 0.012% of bromophenol blue), boiled at 95 °C for 5 min and then separated by 10% SDS-PAGE electrophoresis. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or other:Article Title: Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death Article Snippet: Imaging:Article Title: Escherichia coli Subtilase Cytotoxin Induces Apoptosis Regulated by Host Bcl-2 Family Proteins Bax/Bak ▿ Article Snippet: Anti-Puma (catalog no. P-4618) and Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients Article Snippet: Equal loading and transfer were assessed by reprobing the blots with Protein Concentration:Article Title: Effects of Balance and Gait Training on the Recovery of the Motor Function in an Animal Model of Parkinson's Disease Article Snippet: The samples were centrifuged for 20 min at 15,000 rpm to obtain supernatant, and the protein concentration in the solution was quantified using Bradford method (Bio-Rad protein assay). .. To confirm if the equivalent protein, Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study Article Snippet: Sample lysates were centrifuged at 15,000 rpm for 20 min at 4 °C and protein concentration in the supernatants was determined by the bicinchoninic acid (BCA; Pierce, Rockford, IL, USA) microplate method. .. The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti Binding Assay:Article Title: Effects of Balance and Gait Training on the Recovery of the Motor Function in an Animal Model of Parkinson's Disease Article Snippet: Thereafter, the gel was transferred to a nitrocellulose membrane (Whatman GmbH, Dassel, Germany) and underwent reacted with TBX and 5% non fat dried milk for 1 hr at 4°C to prevent non-specific binding with the antibody. .. To confirm if the equivalent protein, Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke Article Snippet: After electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes and nonspecific binding was blocked with 5% nonfat dry milk in tris-buffered saline and Tween 20. .. After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the Nucleic Acid Electrophoresis:Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: Protein extracts from cultured hippocampal neurons or hippocampal homogenates were diluted in 6× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer ((0.5 M Tris, 0.4% SDS, pH 6.8), 30% glycerol, 10% SDS, 0.6 M dithiothreitol, and 0.012% of bromophenol blue), boiled at 95 °C for 5 min and then separated by 10% SDS-PAGE electrophoresis. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or Isolation:Article Title: LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence Article Snippet: .. Protein was isolated from cultured cortical neurons and western blot analyses performed as previously described, with antibodies directed toward cleaved caspase-3 (Cell Signaling, Cat No. 9664S), Rb (PharMingen, Cat No. 554136), Cyclin A2 (Abcam, Cat No. ab7965), PCNA (Millpore, Cat No. MAB424) and Protein Extraction:Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function Article Snippet: Paragraph title: Protein extraction and quantification ... After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Blocking Assay:Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function Article Snippet: .. After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Article Title: Transcriptomic analysis reveals sex-specific differences in the expression of Dcl1 and Fis1 genes in the radio-adaptive response of thymocytes to TRP53-mediated apoptosis Article Snippet: After blocking, membranes were incubated with each primary antibody in either 5 % w/v BSA or nonfat dry milk, 1× TBS, 0.1 % Tween 20 overnight at 4 °C. .. For Chloramphenicol Acetyltransferase Assay:Article Title: High fat diet increases melanoma cell growth in the bone marrow by inducing osteopontin and interleukin 6 Article Snippet: .. Following protein transfer into 0.45 μM nitrocellulose (NC) membrane (BD, Cat.#1620115), membranes were blocked with 5% skim-milk in TBS-T buffer (0.05%) for 2 h. Then, membranes that washed and were probed using the following antibodies: Nuclear factor kappa B (NF-κB) p65 (Cell signaling Technology, Inc. Cat.#8242; 1:500 dilutions), phospho-NF-κB p65 (Ser468) (Cell signaling Technology, Inc. Cat.#3039; 1:500 dilutions) and Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma Article Snippet: .. Protein detection was performed using anti-NELFE (Abcam, cat# ab170104), Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Catab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or SDS Page:Article Title: Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells Article Snippet: Western blot analysis Proteins from the same volume of each fraction (200 to 250 μl) were concentrated by TCA precipitation, dissolved in SDS/β-mercaptoethanol loading buffer, and resolved on a 4% to 15% gradient SDS/PAGE gel (Bio-Rad, Hercules, CA). .. Antibodies against H3 (ab1791), DNMT3A (ab2850), and SUV39h1 (ab12405) were purchased from Abcam Inc. (Cambridge, UK); DNMT1 (sc-20701) and DNMT3B (sc-10235) from Santa Cruz Biotech (Santa Cruz, CA); Myc epitope tag (05-724) from Upstate (now Millipore, Billerica, MA) and G9a (G 6919); Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function Article Snippet: 50μg of protein extracts were resolved by 4-15% SDS-PAGE (SDS-PAGE Mini- PROTEAN® TGX™ #456-1086 Biorad) and transferred to 0.2 μm nitrocellulose membranes (Biorad). .. After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: Protein extracts from cultured hippocampal neurons or hippocampal homogenates were diluted in 6× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer ((0.5 M Tris, 0.4% SDS, pH 6.8), 30% glycerol, 10% SDS, 0.6 M dithiothreitol, and 0.012% of bromophenol blue), boiled at 95 °C for 5 min and then separated by 10% SDS-PAGE electrophoresis. .. A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or Plasmid Preparation:Article Title: A polymorphism in the lysyl oxidase propeptide domain accelerates carcinogen-induced cancer Article Snippet: .. Antibodies used were Ki-67 (cloneSP6; Thermo Scientific), LOX-PP (rabbit polyclonal IgG) used as described previously , Software:Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke Article Snippet: After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the Article Title: The Effects of Antecedent Exercise on Motor Function Recovery and Brain-derived Neurotrophic Factor Expression after Focal Cerebral Ischemia in Rats Article Snippet: After incubation with the anti-BDNF primary antibody (sc-546, Santa Cruz, USA), Article Title: Escherichia coli Subtilase Cytotoxin Induces Apoptosis Regulated by Host Bcl-2 Family Proteins Bax/Bak ▿ Article Snippet: Anti-Puma (catalog no. P-4618) and Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients Article Snippet: Equal loading and transfer were assessed by reprobing the blots with Article Title: Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation Article Snippet: A rabbit antibody directed against GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (1:1000; Abcam, Cat#ab9485 RRID:AB_307275) and mouse monoclonal antibodies against α-tubulin (1:20 000; Sigma, Cat#T6074 RRID:AB_477582) or Irradiation:Article Title: Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients Article Snippet: Western blot analysis Nearly confluent nonirradiated and irradiated with 8 Gy fibroblasts were detached by trypsinisation, lyzed in RIPA buffer (10 mM tris-HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100) containing protease inhibitors (2 μ g ml−1 aprotinin, 2 μ g ml−1 leupeptin, 5 μ g ml−1 pepstatin, 1 mM phenylmethane sulphonyl fluoride) for 30 min on ice and subsequently centrifuged 10 min at 500 g . .. Equal loading and transfer were assessed by reprobing the blots with Negative Control:Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study Article Snippet: Western Blotting At the endpoint of the culture, both cell/scaffolds constructs (n = 3) and controls (scaffolds without cells as a negative control, and cells grown in monolayer up to 80% confluence—4 days—as a positive control) were washed in D-PBS and resuspended in a cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and antiproteases 1×, all from Sigma Aldrich). .. The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti Radio Immunoprecipitation:Article Title: Modulating BAP1 expression affects ROS homeostasis, cell motility and mitochondrial function Article Snippet: Cell pellets were lysed in radioimmunoprecipitation assay buffer (RIPA) [100mM Tris-HCl/pH7.5, 0.1M NaCl, 1mM EDTA, 1% Triton, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)], supplemented with NaF, Na3 VO4 , phenylmethanesulfonylfluoride (PMSF) and a cocktail of protease inhibitors (cOmplete©, Roche). .. After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C (BAP1, Santa Cruz sc-4 sc-28383, 1/500; CDH2, Cell signaling #14215, 1/200; β-actin, Lysis:Article Title: Beneficial Effects of Antecedent Exercise Training on Limb Motor Function and Calpain Expression in a Rat Model of Stroke Article Snippet: The tissue was homogenized in freshly prepared lysis buffer (1:10 w/v) and centrifuged at 12,000 × g for 30 min. .. After incubation with the rabbit polyclonal anti-m-calpain antibody (1:1,000 Triple Point Biologics; Forest Grove, OR, USA) and the Article Title: Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study Article Snippet: Western Blotting At the endpoint of the culture, both cell/scaffolds constructs (n = 3) and controls (scaffolds without cells as a negative control, and cells grown in monolayer up to 80% confluence—4 days—as a positive control) were washed in D-PBS and resuspended in a cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and antiproteases 1×, all from Sigma Aldrich). .. The membrane was blocked with 4% dry non-fat milk in 0.1% Tween/Tris Buffer Saline (T-TBS) and incubated overnight at 4 °C with anti Article Title: The Effects of Antecedent Exercise on Motor Function Recovery and Brain-derived Neurotrophic Factor Expression after Focal Cerebral Ischemia in Rats Article Snippet: Tissue was homogenized in freshly prepared lysis buffer (1:10 w/v) and centrifuged at 12,000 ×g for 30 min. .. After incubation with the anti-BDNF primary antibody (sc-546, Santa Cruz, USA), |
