Structured Review

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Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm <t>PBS.</t> The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with <t>RIPA</t> buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).
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1) Product Images from "Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1) *"

Article Title: Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.541102

Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).
Figure Legend Snippet: Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

Techniques Used: Activation Assay, Expressing, Incubation

2) Product Images from "Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells"

Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138849

Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P
Figure Legend Snippet: Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

Techniques Used: Electron Microscopy, Isolation, Western Blot, Cell Culture, Activity Assay

Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.
Figure Legend Snippet: Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

Techniques Used: Derivative Assay, Electron Microscopy, Isolation, Western Blot, Cell Culture, SDS Page, Electrophoresis, Staining, Software

3) Product Images from "α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis"

Article Title: α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis

Journal: Theranostics

doi: 10.7150/thno.24385

Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p
Figure Legend Snippet: Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p

Techniques Used: Expressing, Activity Assay, Electrophoresis, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Luciferase

4) Product Images from "Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins"

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

Journal: Oncotarget

doi: 10.18632/oncotarget.8072

Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.
Figure Legend Snippet: Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

Techniques Used: In Vivo, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Plasmid Preparation, Mutagenesis, Transfection

Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.
Figure Legend Snippet: Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

Techniques Used: Incubation, Immunoprecipitation, Transfection, Western Blot

5) Product Images from "Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3"

Article Title: Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022290

An intact SMYD3 MYND domain is required for association with N-CoR and for transcriptional repression. (A) N-CoR co-immunoprecipitates with wildtype SMYD3 but not with SMYD3 MYND domain point mutant C49/S. 293T cells were co-transfected with N-CoR, N-terminal myc-tagged SMYD3 constructs indicated, and with empty vector (vector). 48 hours post-transfection, whole cell RIPA lysates (WCL) were prepared. Fractions of the lysates were subjected to anti-N-CoR co-immunoprecipitation and the remaining 50% served as input. Western analysis was performed with anti-myc antibodies. Myc-SMYD1B, previously shown to interact with N-CoR served as a positive control. (B) Schematic of GAL4-DNA binding domain (DBD) and GAL4-fusion constructs for wild type (GAL4-SMYD3) and MYND domain-mutated (GAL4-SMYD3-C49/S) two hybrid transcription assays. X denotes the location of the C49/S mutation. (C) GAL4-SMYD3 but not GAL4-SMYD3-C49/S represses transcription of a GAL4-UAS containing luciferase reporter. 293T cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 µg) together with GAL4-DBD, or with 1 or 2 µg (indicated as 1X or 2X) of GAL4-SMYD3 (black bars) or GAL4-SMYD3-C49/S (red bars). Transfection efficiencies were normalized to co-transfected renilla luciferase, and percent GAL4 activity was determined in relation to GAL4-DBD set at 100%.
Figure Legend Snippet: An intact SMYD3 MYND domain is required for association with N-CoR and for transcriptional repression. (A) N-CoR co-immunoprecipitates with wildtype SMYD3 but not with SMYD3 MYND domain point mutant C49/S. 293T cells were co-transfected with N-CoR, N-terminal myc-tagged SMYD3 constructs indicated, and with empty vector (vector). 48 hours post-transfection, whole cell RIPA lysates (WCL) were prepared. Fractions of the lysates were subjected to anti-N-CoR co-immunoprecipitation and the remaining 50% served as input. Western analysis was performed with anti-myc antibodies. Myc-SMYD1B, previously shown to interact with N-CoR served as a positive control. (B) Schematic of GAL4-DNA binding domain (DBD) and GAL4-fusion constructs for wild type (GAL4-SMYD3) and MYND domain-mutated (GAL4-SMYD3-C49/S) two hybrid transcription assays. X denotes the location of the C49/S mutation. (C) GAL4-SMYD3 but not GAL4-SMYD3-C49/S represses transcription of a GAL4-UAS containing luciferase reporter. 293T cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 µg) together with GAL4-DBD, or with 1 or 2 µg (indicated as 1X or 2X) of GAL4-SMYD3 (black bars) or GAL4-SMYD3-C49/S (red bars). Transfection efficiencies were normalized to co-transfected renilla luciferase, and percent GAL4 activity was determined in relation to GAL4-DBD set at 100%.

Techniques Used: Mutagenesis, Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Positive Control, Binding Assay, Luciferase, Activity Assay

6) Product Images from "Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes"

Article Title: Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-7-59

Shifts in full length APP patterns induced by the glycosylation inhibitors tunicamycin and brefeldin A in mononuclear phagocyte cultures . Human mononuclear phagocytes were isolated as indicated and left unstimulated on ultra-low attachment plates for 3 days. 6 h, 4 h, 2 h or 0.5 h prior to the lysis of the cells, tunicamycin or brefeldin A were added in a concentration of 10 μg/ml each. Cells were lysed in RIPA buffer and APP expression was analysed by separation on 7.5% SDS-PAGE, subsequent blotting on PVDF-membranes and staining with the 1E8 monoclonal antibody. Staining of β-actin served as a loading control. The right hand side shows a molecular weight standard. Note the slight shift in molecular weight after brefeldin A treatment and the decrease of band APP1 corresponding to mature APP and the increased amounts of the bands APP2 and APP3 after treatment with Tunicamycin.
Figure Legend Snippet: Shifts in full length APP patterns induced by the glycosylation inhibitors tunicamycin and brefeldin A in mononuclear phagocyte cultures . Human mononuclear phagocytes were isolated as indicated and left unstimulated on ultra-low attachment plates for 3 days. 6 h, 4 h, 2 h or 0.5 h prior to the lysis of the cells, tunicamycin or brefeldin A were added in a concentration of 10 μg/ml each. Cells were lysed in RIPA buffer and APP expression was analysed by separation on 7.5% SDS-PAGE, subsequent blotting on PVDF-membranes and staining with the 1E8 monoclonal antibody. Staining of β-actin served as a loading control. The right hand side shows a molecular weight standard. Note the slight shift in molecular weight after brefeldin A treatment and the decrease of band APP1 corresponding to mature APP and the increased amounts of the bands APP2 and APP3 after treatment with Tunicamycin.

Techniques Used: Isolation, Lysis, Concentration Assay, Expressing, SDS Page, Staining, Molecular Weight

7) Product Images from "F3/Contactin-Related Proteins in Helix pomatia Nervous Tissue (HCRPs): Distribution and Function in Neurite Growth and Neurotransmitter Release"

Article Title: F3/Contactin-Related Proteins in Helix pomatia Nervous Tissue (HCRPs): Distribution and Function in Neurite Growth and Neurotransmitter Release

Journal: Journal of neuroscience research

doi: 10.1002/jnr.21539

Molecular heterogeneity of HCRPs. A: Isolation of a HCRP cDNA. The nucleotide sequence of clone 9.1, isolated from a Helix ganglia expression library with the mouse F3/contactin antiserum, is shown. B: Identification of the HCRP mRNA. Total and poly(A) + RNAs from Helix ganglia were probed by Northern blot with a 32 P-labelled 9.1 cDNA probe. A main 6.3-kb chain was detected in both cases. C: N-deglycosylation of HCRPs. Detergent extracts from Helix pomatia ganglia in RIPA buffer were treated with N-glycosidase F and probed with the 24III antiserum. In digested samples (+), the 240-kD band disappeared, and the 200-kD chain become thinner. Minor differences were found for the 90-kD isoform, which appeared as a doublet of closely spaced bands.
Figure Legend Snippet: Molecular heterogeneity of HCRPs. A: Isolation of a HCRP cDNA. The nucleotide sequence of clone 9.1, isolated from a Helix ganglia expression library with the mouse F3/contactin antiserum, is shown. B: Identification of the HCRP mRNA. Total and poly(A) + RNAs from Helix ganglia were probed by Northern blot with a 32 P-labelled 9.1 cDNA probe. A main 6.3-kb chain was detected in both cases. C: N-deglycosylation of HCRPs. Detergent extracts from Helix pomatia ganglia in RIPA buffer were treated with N-glycosidase F and probed with the 24III antiserum. In digested samples (+), the 240-kD band disappeared, and the 200-kD chain become thinner. Minor differences were found for the 90-kD isoform, which appeared as a doublet of closely spaced bands.

Techniques Used: Isolation, Sequencing, Expressing, Northern Blot

8) Product Images from "Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma"

Article Title: Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma

Journal: Scientific Reports

doi: 10.1038/srep21596

USP4 regulated the expression of β-catenin by controlling its protein stability. ( A ) To compare the β-catenin protein stability, PC14PE6 and PC14PE6/LvBr4 cells were treated with cycloheximide (20 μg/mL) and harvested at the indicated times. Whole cell lysates were prepared, and the level of β-catenin protein was determined by western blotting. The stability of β-catenin was assessed by image analysis. ( B ) The expression level of USP4 in PC14PE6 and PC14PE6/LvBr4 cells was examined by western blotting (left panel) and RT-qPCR (right panel). ( C ) To determine whether USP regulates the expression of β-catenin, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The whole cell extract was prepared and the expression level of USP4 and β-catenin was determined by western blotting. ( D,E ) To evaluate the direct interaction between USP4 and β-catenin, whole cell lysates were prepared using RIPA buffer, and equal amounts of protein were incubated with appropriate control IgG or an antibody against USP4 ( D ) or β-catenin ( E ). The level of β-catenin and USP4 in IP materials was assessed by western blotting. ( F ) To check the effect of USP4 silencing on the ubiquitination of β-catenin, PC14PE6 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. Whole cell lysates were prepared using RIPA buffer and incubated with appropriate control IgG and β-catenin antibody. The levels of β-catenin and ubiquitin were assessed by western blotting. ( G ) To compare the protein stability of β-catenin, parental PC14PE6 and brain metastatic PC14PE6/LvBr4 cells were treated with cycloheximide (40 μg/mL) and harvested at the indicated times. The stability of β-catenin was determined as described above. Data are means and standard deviation from more than three independent experiments. * p
Figure Legend Snippet: USP4 regulated the expression of β-catenin by controlling its protein stability. ( A ) To compare the β-catenin protein stability, PC14PE6 and PC14PE6/LvBr4 cells were treated with cycloheximide (20 μg/mL) and harvested at the indicated times. Whole cell lysates were prepared, and the level of β-catenin protein was determined by western blotting. The stability of β-catenin was assessed by image analysis. ( B ) The expression level of USP4 in PC14PE6 and PC14PE6/LvBr4 cells was examined by western blotting (left panel) and RT-qPCR (right panel). ( C ) To determine whether USP regulates the expression of β-catenin, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The whole cell extract was prepared and the expression level of USP4 and β-catenin was determined by western blotting. ( D,E ) To evaluate the direct interaction between USP4 and β-catenin, whole cell lysates were prepared using RIPA buffer, and equal amounts of protein were incubated with appropriate control IgG or an antibody against USP4 ( D ) or β-catenin ( E ). The level of β-catenin and USP4 in IP materials was assessed by western blotting. ( F ) To check the effect of USP4 silencing on the ubiquitination of β-catenin, PC14PE6 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. Whole cell lysates were prepared using RIPA buffer and incubated with appropriate control IgG and β-catenin antibody. The levels of β-catenin and ubiquitin were assessed by western blotting. ( G ) To compare the protein stability of β-catenin, parental PC14PE6 and brain metastatic PC14PE6/LvBr4 cells were treated with cycloheximide (40 μg/mL) and harvested at the indicated times. The stability of β-catenin was determined as described above. Data are means and standard deviation from more than three independent experiments. * p

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Incubation, Standard Deviation

9) Product Images from "Involvement of Osteocytes in the Action of Pasteurella multocida Toxin"

Article Title: Involvement of Osteocytes in the Action of Pasteurella multocida Toxin

Journal: Toxins

doi: 10.3390/toxins10080328

MLO-Y4 cells are susceptible to PMT. Deamidation of heterotrimeric G proteins in MLO-Y4 cells is shown by immunoblot ( A ) or immunofluorescence ( B ) analyses. ( A ) Cells were intoxicated overnight with PMT, PMT C1165S (1 nM each) or not (con) and the deamidation in lysates was detected in immunoblot analysis using the deamidation-specific anti-GαQE antibody. A representative immunoblot from 3 independent experiments is shown. Tubulin was used as loading control. ( B ) After intoxication, deamidation of Gα was determined by immunofluorescence using a deamidation-specific anti-GαQE antibody (green) and 565-phalloidin (red). Immunofluorescence was performed as described in “Materials and Methods”. Microscopy images were captured using the same exposure time and were edited in the same manner (scale bar = 10 µm). Representable pictures from one of 3 independent experiments are shown. ( C ) Morphological characterization of MLO-Y4 cells after treatment with PMT (1 nM) for 24 h. Actin is stained with 565-phalloidin (red) and nuclei are depicted in blue. (Scale bar indicates 20 µm). Representative images from one of 3 independent experiments are shown ( D ) For the RhoC activation assay, MLO-Y4 were serum starved overnight. Cells were incubated for 4 h with PMT, PMT C1165S (1 nM each) or not (con). Cells were lysed and rhotekin pulldown assay was performed. Only PMT led to a pulldown of activated RhoC. Equal loading was verified by probing RhoC in whole cell lysates. ( E/F ) Immunoblot analysis of pERK 1/2 and pAkt. MLO-Y4 were serum starved overnight. Cells were then incubated with PMT, PMT C1165S (1 nM each) or not (con) for 4 h and lysed with RIPA buffer. Tubulin was used as loading control. Shown are mean values of at least three independent experiments ( n = 3; ±SEM). The corresponding panels show representative immunoblots. Statistical analyses were performed using one-way ANOVA. *** p
Figure Legend Snippet: MLO-Y4 cells are susceptible to PMT. Deamidation of heterotrimeric G proteins in MLO-Y4 cells is shown by immunoblot ( A ) or immunofluorescence ( B ) analyses. ( A ) Cells were intoxicated overnight with PMT, PMT C1165S (1 nM each) or not (con) and the deamidation in lysates was detected in immunoblot analysis using the deamidation-specific anti-GαQE antibody. A representative immunoblot from 3 independent experiments is shown. Tubulin was used as loading control. ( B ) After intoxication, deamidation of Gα was determined by immunofluorescence using a deamidation-specific anti-GαQE antibody (green) and 565-phalloidin (red). Immunofluorescence was performed as described in “Materials and Methods”. Microscopy images were captured using the same exposure time and were edited in the same manner (scale bar = 10 µm). Representable pictures from one of 3 independent experiments are shown. ( C ) Morphological characterization of MLO-Y4 cells after treatment with PMT (1 nM) for 24 h. Actin is stained with 565-phalloidin (red) and nuclei are depicted in blue. (Scale bar indicates 20 µm). Representative images from one of 3 independent experiments are shown ( D ) For the RhoC activation assay, MLO-Y4 were serum starved overnight. Cells were incubated for 4 h with PMT, PMT C1165S (1 nM each) or not (con). Cells were lysed and rhotekin pulldown assay was performed. Only PMT led to a pulldown of activated RhoC. Equal loading was verified by probing RhoC in whole cell lysates. ( E/F ) Immunoblot analysis of pERK 1/2 and pAkt. MLO-Y4 were serum starved overnight. Cells were then incubated with PMT, PMT C1165S (1 nM each) or not (con) for 4 h and lysed with RIPA buffer. Tubulin was used as loading control. Shown are mean values of at least three independent experiments ( n = 3; ±SEM). The corresponding panels show representative immunoblots. Statistical analyses were performed using one-way ANOVA. *** p

Techniques Used: Immunofluorescence, Microscopy, Staining, Activation Assay, Incubation, Western Blot

10) Product Images from "Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation"

Article Title: Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation

Journal: Journal of Molecular Histology

doi: 10.1007/s10735-012-9415-1

Electron-microscoipic observations of hormone-refractory-prostate cancer cell and prostate cancer cell-derived microvesicles (PCa-MVs) ( a ), analysis of microvesicle profile from human prostate cancer PC3 cells ( b ) and biochemical study of MVs from PC3 and DU145 cells ( c ). a Both hormone-refractory-PCa cell lines PC3 cells and DU145 cells shed microvesicles from their cell membrane. The black arrowheads indicate PCa-MVs. The right photos of PC3 and DU145 cells show enlarged views of rectangular areas in the left photos. b MVs were isolated from PC3 cells and measured with NTA. For preparation of MVs from PC3 cells, the medium from either source was centrifuged at 1,500× g for 10 min to remove cells and other debris. These supernatants were then centrifuged at 250,000× g for 3 h at 4 °C. The centrifuged-microvesicles were resuspended in serum-free α-MEM and then filtered (0.45 μm). The microvesicle samples after passage through the 1st filter (0.22 μm) of an ExoMir kit were used for analysis. The Nanosight LM10 nanoparticle characterization system (NanoSight, NanoSight Ltd, UK) equipped with a blue laser (638 nm) illumination was used for real-time characterization of the vesicles. The results were presented at the average value of 2 independent experiments. The number of microvesicles (E6 particles/ml) and the size distribution (particle diameter, nm) are shown on the y axis and x axis, respectively. c Western blots of MV-related proteins, i.e., TSG101, CD9, and CD81. MVs were prepared from the culture medium by ultracentrifugation (250,000× g for 3 h, 4 °C). The microvesicles were further filtered (0.45 μm) and then resuspended in RIPA buffer containing the complete protease inhibitor cocktail. Three micrograms protein per lane was loaded. The relative expression levels of the MV-related proteins indicate a specificity for MVs
Figure Legend Snippet: Electron-microscoipic observations of hormone-refractory-prostate cancer cell and prostate cancer cell-derived microvesicles (PCa-MVs) ( a ), analysis of microvesicle profile from human prostate cancer PC3 cells ( b ) and biochemical study of MVs from PC3 and DU145 cells ( c ). a Both hormone-refractory-PCa cell lines PC3 cells and DU145 cells shed microvesicles from their cell membrane. The black arrowheads indicate PCa-MVs. The right photos of PC3 and DU145 cells show enlarged views of rectangular areas in the left photos. b MVs were isolated from PC3 cells and measured with NTA. For preparation of MVs from PC3 cells, the medium from either source was centrifuged at 1,500× g for 10 min to remove cells and other debris. These supernatants were then centrifuged at 250,000× g for 3 h at 4 °C. The centrifuged-microvesicles were resuspended in serum-free α-MEM and then filtered (0.45 μm). The microvesicle samples after passage through the 1st filter (0.22 μm) of an ExoMir kit were used for analysis. The Nanosight LM10 nanoparticle characterization system (NanoSight, NanoSight Ltd, UK) equipped with a blue laser (638 nm) illumination was used for real-time characterization of the vesicles. The results were presented at the average value of 2 independent experiments. The number of microvesicles (E6 particles/ml) and the size distribution (particle diameter, nm) are shown on the y axis and x axis, respectively. c Western blots of MV-related proteins, i.e., TSG101, CD9, and CD81. MVs were prepared from the culture medium by ultracentrifugation (250,000× g for 3 h, 4 °C). The microvesicles were further filtered (0.45 μm) and then resuspended in RIPA buffer containing the complete protease inhibitor cocktail. Three micrograms protein per lane was loaded. The relative expression levels of the MV-related proteins indicate a specificity for MVs

Techniques Used: Derivative Assay, Isolation, Western Blot, Protease Inhibitor, Expressing

11) Product Images from "Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease"

Article Title: Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

Journal: Nature genetics

doi: 10.1038/ng.2901

Expression of TM6SF2 in cultured hepatocytes. ( a ) Plasmids encoding wild-type and mutant human TM6SF2 were expressed in HuH7 cells. Two days after transfection, the TM6SF2 mRNA levels were measured using Real-Time PCR (left). The cells were harvested and solubilized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH=8). Quantitative immunoblotting was performed using a LI-COR Odyssey infrared imaging system as described in the Methods (right). The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments. V, vector. ( b ) Recombinant wild-type hTM6SF2 was expressed in Hepa1c1c7 cells. After two days, the cells were fractionated and subjected to immunoblotting as described in the Methods. C, cytosol; M, membranes; LD, lipid droplets; L, whole cell lysate. The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments.
Figure Legend Snippet: Expression of TM6SF2 in cultured hepatocytes. ( a ) Plasmids encoding wild-type and mutant human TM6SF2 were expressed in HuH7 cells. Two days after transfection, the TM6SF2 mRNA levels were measured using Real-Time PCR (left). The cells were harvested and solubilized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH=8). Quantitative immunoblotting was performed using a LI-COR Odyssey infrared imaging system as described in the Methods (right). The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments. V, vector. ( b ) Recombinant wild-type hTM6SF2 was expressed in Hepa1c1c7 cells. After two days, the cells were fractionated and subjected to immunoblotting as described in the Methods. C, cytosol; M, membranes; LD, lipid droplets; L, whole cell lysate. The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments.

Techniques Used: Expressing, Cell Culture, Mutagenesis, Transfection, Real-time Polymerase Chain Reaction, Imaging, Plasmid Preparation, Recombinant

12) Product Images from "Ubiquitin-dependent regulation of Cdc42 by XIAP"

Article Title: Ubiquitin-dependent regulation of Cdc42 by XIAP

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.305

( a ) Interaction between XIAP and different Cdc42 mutants was tested via an in vitro GST Pulldown assay. With GST protein as a control, two different concentrations of cleaved Cdc42 mutants were used to test the binding to GST tagged XIAP ( b ) In vitro ubiquitination of Cdc42 by XIAP. Purified recombinant Cdc42Q61L was subjected to in vitro ubiquitination by XIAP and cIAP1 recombinant proteins (protocol described in Materials and Methods). ( c ) HeLa cells were transfected with XIAP siRNA for 48 h and treated with MG132 for 6 h. The cells were lysed in RIPA buffer and His-TUBE immobilized on Ni-NTA beads were employed to enrich the ubiquitinated proteins. The samples were loaded onto a gel and the presence of Cdc42 was monitored by immunoblots. The efficiency of XIAP knockdown was tested in the lysates control. * denotes an unspecific band. ( d ) Gel slices of the in vitro ubiquitination reaction were subjected to mass spectrometric analysis to determine the Lysine(s) responsible for the ubiquitination. Inset shows the gel slices taken for the analysis as well as a comparison between the sequences of Rac1 and Cdc42 ( e ) Mouse embryonic fibroblasts (MEFs) were cultured and lysed to check for Cdc42 levels. Control MEFs and XIAP knockout MEFs stably complemented with different XIAP constructs were used for this experiment. * denotes lower exposure of Cdc42
Figure Legend Snippet: ( a ) Interaction between XIAP and different Cdc42 mutants was tested via an in vitro GST Pulldown assay. With GST protein as a control, two different concentrations of cleaved Cdc42 mutants were used to test the binding to GST tagged XIAP ( b ) In vitro ubiquitination of Cdc42 by XIAP. Purified recombinant Cdc42Q61L was subjected to in vitro ubiquitination by XIAP and cIAP1 recombinant proteins (protocol described in Materials and Methods). ( c ) HeLa cells were transfected with XIAP siRNA for 48 h and treated with MG132 for 6 h. The cells were lysed in RIPA buffer and His-TUBE immobilized on Ni-NTA beads were employed to enrich the ubiquitinated proteins. The samples were loaded onto a gel and the presence of Cdc42 was monitored by immunoblots. The efficiency of XIAP knockdown was tested in the lysates control. * denotes an unspecific band. ( d ) Gel slices of the in vitro ubiquitination reaction were subjected to mass spectrometric analysis to determine the Lysine(s) responsible for the ubiquitination. Inset shows the gel slices taken for the analysis as well as a comparison between the sequences of Rac1 and Cdc42 ( e ) Mouse embryonic fibroblasts (MEFs) were cultured and lysed to check for Cdc42 levels. Control MEFs and XIAP knockout MEFs stably complemented with different XIAP constructs were used for this experiment. * denotes lower exposure of Cdc42

Techniques Used: In Vitro, GST Pulldown Assay, Binding Assay, Purification, Recombinant, Transfection, Western Blot, Cell Culture, Knock-Out, Stable Transfection, Construct

13) Product Images from "SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis"

Article Title: SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

Journal: Molecular Cancer

doi: 10.1186/s12943-017-0724-6

SUMOylation of KHSRP is involved in tumorigenesis. a KHSRP-K87R downregulates the anchorage-independent growth in DU145 stable cell lines. In soft agar colony forming assays, stable cell lines DU145 shRNA control, shKHSRP, shKHSRP-KHSRP-WT or shKHSRP-KHSRP-K87R were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 2 × 10 3 cells/well and layered onto the base. The photographs were taken 21 days later and the number of colonies was scored. b KHSRP-K87R downregulates the migration ability in DU145 stable cell lines. The RTCA migration assay was performed to detect the migration ability in above stable DU145 cell lines with xCELLigene RTCA-DP instrument. The kinetic cell index of their migration was recorded every 15 min for 24 h (left panel) and the relative slope value was calculated (right panel). c KHSRP-K87R downregulates the invasive ability in above stable DU145 cell lines. The 3D–culture assay was performed to detect the invasive ability of DU145 stable cell lines. The photos were taken at day 7. The first image was taken under the white light, and the green signals indicates the expression of GFP (Green Fluorescent Protein) in the plasmid CD513B-HA-KHSRP. d KHSRP-K87R downregulates the aggressive ability in DU145 stable cell lines in vasculogenic mimicry (VM) assay. VM assay was performed to detect the aggressive ability in above stable DU145 cell lines. The photos were taken 20 h later. Scale: 500 μm. Independent experiments ( a - d ) were repeated three times. e KHSRP-K87R suppresses xenograft tumor growth in vivo. 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell lines (2.5 × 10 6 cells/each) expressing the shRNA control in the left back and shKHSRP in the right back, respectively. Another 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell line expressing shKHSRP-KHSRP-WT in the left back and shKHSRP-KHSRP-K87R in the right back, respectively. Mice were sacrificed 5 weeks later, and tumors were dissected (upper panel) and assessed by weight (low panel). ( a - d ) f KHSRP SUMOylation could be detected in tumors of nude mice. The tumors of nude mice, which was chosen from the groups of shKHSRP, shKHSRP-KHSRP-WT or -K87R, were lysed in NEM-RIPA as described in the Methods. The proteins was immunoprecipitated by anti-SUMO1 antibody, Western blotting was detected with anti-HA antibody
Figure Legend Snippet: SUMOylation of KHSRP is involved in tumorigenesis. a KHSRP-K87R downregulates the anchorage-independent growth in DU145 stable cell lines. In soft agar colony forming assays, stable cell lines DU145 shRNA control, shKHSRP, shKHSRP-KHSRP-WT or shKHSRP-KHSRP-K87R were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 2 × 10 3 cells/well and layered onto the base. The photographs were taken 21 days later and the number of colonies was scored. b KHSRP-K87R downregulates the migration ability in DU145 stable cell lines. The RTCA migration assay was performed to detect the migration ability in above stable DU145 cell lines with xCELLigene RTCA-DP instrument. The kinetic cell index of their migration was recorded every 15 min for 24 h (left panel) and the relative slope value was calculated (right panel). c KHSRP-K87R downregulates the invasive ability in above stable DU145 cell lines. The 3D–culture assay was performed to detect the invasive ability of DU145 stable cell lines. The photos were taken at day 7. The first image was taken under the white light, and the green signals indicates the expression of GFP (Green Fluorescent Protein) in the plasmid CD513B-HA-KHSRP. d KHSRP-K87R downregulates the aggressive ability in DU145 stable cell lines in vasculogenic mimicry (VM) assay. VM assay was performed to detect the aggressive ability in above stable DU145 cell lines. The photos were taken 20 h later. Scale: 500 μm. Independent experiments ( a - d ) were repeated three times. e KHSRP-K87R suppresses xenograft tumor growth in vivo. 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell lines (2.5 × 10 6 cells/each) expressing the shRNA control in the left back and shKHSRP in the right back, respectively. Another 5 male BALB/c nude mice were injected subcutaneously with stable DU145 cell line expressing shKHSRP-KHSRP-WT in the left back and shKHSRP-KHSRP-K87R in the right back, respectively. Mice were sacrificed 5 weeks later, and tumors were dissected (upper panel) and assessed by weight (low panel). ( a - d ) f KHSRP SUMOylation could be detected in tumors of nude mice. The tumors of nude mice, which was chosen from the groups of shKHSRP, shKHSRP-KHSRP-WT or -K87R, were lysed in NEM-RIPA as described in the Methods. The proteins was immunoprecipitated by anti-SUMO1 antibody, Western blotting was detected with anti-HA antibody

Techniques Used: Stable Transfection, shRNA, Migration, Expressing, Plasmid Preparation, VM Assay, In Vivo, Mouse Assay, Injection, Immunoprecipitation, Western Blot

14) Product Images from "Identification of Daxx interacting with p73, one of the p53 family, and its regulation of p53 activity by competitive interaction with PML"

Article Title: Identification of Daxx interacting with p73, one of the p53 family, and its regulation of p53 activity by competitive interaction with PML

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkg741

In vivo interaction assay by immunoprecipitation. Transfection conditions in p53-null Saos2 cells were as follows: ( A ) GFP–Daxx and empty HA-pcDNA3 vector, HA-p73α or HA-p53; ( B , left) p73α without tag (in pcDNA3) and empty GFP vector or GFP–Daxx; (B, right) Daxx and empty pcDNA3 or p73α; ( C , left) p53 in pcDNA3 and empty GFP vector or GFP–Daxx; (C, right) Daxx and empty pcDNA3 or p53. After transfections, cells were lysed in RIPA buffer (250 mM NaCl, 50 mM HEPES pH 7.0, 0.1% NP40 and 5 mM EDTA) supplemented with protease inhibitors and pre-cleared by incubating with protein A–Sepharose for 1 h. Immuno precipitation was carried out with anti-HA, anti-Daxx or anti-GFP antibody. After resolution by 10% SDS–PAGE, western blotting was carried out using anti-GFP (A), anti-p73α (B) and anti-p53 (C) antibodies. A band in the control sample (C, right panel, Daxx alone) corresponds to the heavy chain of anti-Daxx antibody that can be detected by western blotting. The band is close to that of p53 because of running the gel for a short time.
Figure Legend Snippet: In vivo interaction assay by immunoprecipitation. Transfection conditions in p53-null Saos2 cells were as follows: ( A ) GFP–Daxx and empty HA-pcDNA3 vector, HA-p73α or HA-p53; ( B , left) p73α without tag (in pcDNA3) and empty GFP vector or GFP–Daxx; (B, right) Daxx and empty pcDNA3 or p73α; ( C , left) p53 in pcDNA3 and empty GFP vector or GFP–Daxx; (C, right) Daxx and empty pcDNA3 or p53. After transfections, cells were lysed in RIPA buffer (250 mM NaCl, 50 mM HEPES pH 7.0, 0.1% NP40 and 5 mM EDTA) supplemented with protease inhibitors and pre-cleared by incubating with protein A–Sepharose for 1 h. Immuno precipitation was carried out with anti-HA, anti-Daxx or anti-GFP antibody. After resolution by 10% SDS–PAGE, western blotting was carried out using anti-GFP (A), anti-p73α (B) and anti-p53 (C) antibodies. A band in the control sample (C, right panel, Daxx alone) corresponds to the heavy chain of anti-Daxx antibody that can be detected by western blotting. The band is close to that of p53 because of running the gel for a short time.

Techniques Used: In Vivo, Immunoprecipitation, Transfection, Plasmid Preparation, SDS Page, Western Blot

15) Product Images from "α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis"

Article Title: α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis

Journal: Theranostics

doi: 10.7150/thno.24385

Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p
Figure Legend Snippet: Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p

Techniques Used: Expressing, Activity Assay, Electrophoresis, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Luciferase

16) Product Images from "Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane"

Article Title: Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.444034

Reduction of the level of phosphorylated MARCKS by PSA. A and B , primary hippocampal neurons were incubated without ( control ) or with chondroitin sulfate ( CS ) or colominic acid ( PSA ). A , neurons were lysed with RIPA buffer and probed by Western blot (
Figure Legend Snippet: Reduction of the level of phosphorylated MARCKS by PSA. A and B , primary hippocampal neurons were incubated without ( control ) or with chondroitin sulfate ( CS ) or colominic acid ( PSA ). A , neurons were lysed with RIPA buffer and probed by Western blot (

Techniques Used: Incubation, Western Blot

17) Product Images from "Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells"

Article Title: Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells

Journal: eLife

doi: 10.7554/eLife.46667

The effect of KPT-330 on gene expression, and binding of CRM1 to HOX regions in MEGAL and HEL cells. ( A-B ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. ( A ) MEGAL and ( B ) HEL cells were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting using anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( C ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in MEGAL and HEL cells treated without or with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( D ) The data in ( C ) were reanalyzed to obtain a ratio by comparing with the values for DMSO-treated samples. Note that low expression genes ( HOXA9 in HEL and HOXB4 in MEGAL) were omitted in ( D ). Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; *p
Figure Legend Snippet: The effect of KPT-330 on gene expression, and binding of CRM1 to HOX regions in MEGAL and HEL cells. ( A-B ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. ( A ) MEGAL and ( B ) HEL cells were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting using anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( C ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in MEGAL and HEL cells treated without or with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( D ) The data in ( C ) were reanalyzed to obtain a ratio by comparing with the values for DMSO-treated samples. Note that low expression genes ( HOXA9 in HEL and HOXB4 in MEGAL) were omitted in ( D ). Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; *p

Techniques Used: Expressing, Binding Assay, Cell Culture, Real-time Polymerase Chain Reaction

Protein expression and CRM1 binding profiles in HL60 cells. ( A ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. LOUCY and HL60 cell lines were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting with anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( B–D ) CRM1 ChIP-seq for HL60 cells was performed as described in Figure 1 . ( B : whole genome; C : whole chromosome 17 and HOXB cluster, D : whole chromosome seven and HOXA cluster).
Figure Legend Snippet: Protein expression and CRM1 binding profiles in HL60 cells. ( A ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. LOUCY and HL60 cell lines were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting with anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( B–D ) CRM1 ChIP-seq for HL60 cells was performed as described in Figure 1 . ( B : whole genome; C : whole chromosome 17 and HOXB cluster, D : whole chromosome seven and HOXA cluster).

Techniques Used: Expressing, Binding Assay, Cell Culture, Chromatin Immunoprecipitation

The effect of KPT-330 on the protein and HOX gene expression levels in OCI-AML3 cells. ( A ) Protein expression of CRM1, NPM1c and GAPDH in OCI-AML3 cells cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. The cell lysates were prepared in RIPA buffer and analyzed by immunoblotting using anti-CRM1, anti-NPM1c, or anti-GAPDH antibodies. ( B ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in OCI-AML3 cells treated with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( C ) The data in ( B ) were reanalyzed to obtain a ratio by comparing the values of the KPT-330-treated samples with the values for DMSO-treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; **p
Figure Legend Snippet: The effect of KPT-330 on the protein and HOX gene expression levels in OCI-AML3 cells. ( A ) Protein expression of CRM1, NPM1c and GAPDH in OCI-AML3 cells cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. The cell lysates were prepared in RIPA buffer and analyzed by immunoblotting using anti-CRM1, anti-NPM1c, or anti-GAPDH antibodies. ( B ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in OCI-AML3 cells treated with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( C ) The data in ( B ) were reanalyzed to obtain a ratio by comparing the values of the KPT-330-treated samples with the values for DMSO-treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; **p

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

18) Product Images from "Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity"

Article Title: Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity

Journal: Retrovirology

doi: 10.1186/1742-4690-4-69

Western blot analysis of transfected HeLa-tat cell and precipitated viruses . HeLa-tat cells were transfected with the plasmids indicated using the non-liposomal transfection reagent. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Particles released into the culture supernatant were also clarified and filtered of cell debris and precipitated with Viraffinity (CPG) as recommended by the manufacturer. Denatured cell (A and B) and viral lysates (C) were then separated by SDS-PAGE, transferred onto a nitrocellulose membrane and detected with a rabbit anti-HIV glycoprotein (A), a pool of anti-CAp24 and anti-calnexin (B), and anti-CAp24 (C) antibodies. The positions of specific viral proteins are indicated to the left and the numbers to the right depict positions of molecular mass markers (in kDa). NT, a mock control; WT, wild type; and D51N, D51E, and D51Q are the three CAp24 mutants.
Figure Legend Snippet: Western blot analysis of transfected HeLa-tat cell and precipitated viruses . HeLa-tat cells were transfected with the plasmids indicated using the non-liposomal transfection reagent. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Particles released into the culture supernatant were also clarified and filtered of cell debris and precipitated with Viraffinity (CPG) as recommended by the manufacturer. Denatured cell (A and B) and viral lysates (C) were then separated by SDS-PAGE, transferred onto a nitrocellulose membrane and detected with a rabbit anti-HIV glycoprotein (A), a pool of anti-CAp24 and anti-calnexin (B), and anti-CAp24 (C) antibodies. The positions of specific viral proteins are indicated to the left and the numbers to the right depict positions of molecular mass markers (in kDa). NT, a mock control; WT, wild type; and D51N, D51E, and D51Q are the three CAp24 mutants.

Techniques Used: Western Blot, Transfection, SDS Page

Western blot analysis of cell-type dependent expression of HIV-1 proteins . HeLa-tat III, 293T and COS7 cells were transfected as described above with mutant and wild type proviral DNA constructs. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Denatured cell lysates were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with a pool of two HIV-1 positive sera (A), rabbit anti-cyclophilin A (B), and anti-calnexin (C) antibodies. Positions of specific viral and cellular proteins are indicated on the right.
Figure Legend Snippet: Western blot analysis of cell-type dependent expression of HIV-1 proteins . HeLa-tat III, 293T and COS7 cells were transfected as described above with mutant and wild type proviral DNA constructs. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Denatured cell lysates were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with a pool of two HIV-1 positive sera (A), rabbit anti-cyclophilin A (B), and anti-calnexin (C) antibodies. Positions of specific viral and cellular proteins are indicated on the right.

Techniques Used: Western Blot, Expressing, Transfection, Mutagenesis, Construct, SDS Page

19) Product Images from "Chemoresistance to Cancer Treatment: Benzo-α-Pyrene as Friend or Foe?"

Article Title: Chemoresistance to Cancer Treatment: Benzo-α-Pyrene as Friend or Foe?

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23040930

Benzo-α-pyrene reverse the effect of drugs on Akt and MEK-ERK signaling pathways in WHCO1 cells. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, and 10 µM BaP for 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, 5-fluorouracil, and BaP; ( B ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with 5-fluorouracil, paclitaxel, and BaP; ( C ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, paclitaxel, and BaP.
Figure Legend Snippet: Benzo-α-pyrene reverse the effect of drugs on Akt and MEK-ERK signaling pathways in WHCO1 cells. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, and 10 µM BaP for 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, 5-fluorouracil, and BaP; ( B ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with 5-fluorouracil, paclitaxel, and BaP; ( C ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, paclitaxel, and BaP.

Techniques Used: BIA-KA

Benzo-α-pyrene differentially influence the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 in response to chemotherapeutic drugs. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 3.5 µM 5-FU, 4.2 µM cisplatin, 2 µM paclitaxel, and 10 µM BaP for 6, 12, and 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with 5-FU and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( B ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with cisplatin and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( C ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with paclitaxel and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies. GAPDH was used as a loading control.
Figure Legend Snippet: Benzo-α-pyrene differentially influence the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 in response to chemotherapeutic drugs. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 3.5 µM 5-FU, 4.2 µM cisplatin, 2 µM paclitaxel, and 10 µM BaP for 6, 12, and 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with 5-FU and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( B ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with cisplatin and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( C ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with paclitaxel and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies. GAPDH was used as a loading control.

Techniques Used: Expressing, BIA-KA

20) Product Images from "Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA"

Article Title: Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

Journal: Journal of Biomedical Research

doi: 10.7555/JBR.26.20120057

Characterization of 6A2. A: The specificity of 6A2 was confirmed by Western blotting assays. Lane 1: pre-immune mouse serum, lane 2: purified 6A2, lane 3: final boost mouse serum. B: Immunoprecipitation of 6A2. Lane 1: a positive control (0.2 µg rGP73 protein), lane 2: a negative control (6A2 + RIPA buffer), lane 3: PC-3 cell lysate, lane 4: MCF-7 cell lysate. IHC staining using 6A2 on sections of HCC (C) and BPH (D) tissue (×400). GP73 staining was mainly detected in the perinuclear region of HCC cells and in the luminal side of prostatic gland epithelia (BPH).
Figure Legend Snippet: Characterization of 6A2. A: The specificity of 6A2 was confirmed by Western blotting assays. Lane 1: pre-immune mouse serum, lane 2: purified 6A2, lane 3: final boost mouse serum. B: Immunoprecipitation of 6A2. Lane 1: a positive control (0.2 µg rGP73 protein), lane 2: a negative control (6A2 + RIPA buffer), lane 3: PC-3 cell lysate, lane 4: MCF-7 cell lysate. IHC staining using 6A2 on sections of HCC (C) and BPH (D) tissue (×400). GP73 staining was mainly detected in the perinuclear region of HCC cells and in the luminal side of prostatic gland epithelia (BPH).

Techniques Used: Western Blot, Purification, Immunoprecipitation, Positive Control, Negative Control, Immunohistochemistry, Staining

21) Product Images from "Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease"

Article Title: Tauopathic Changes in the Striatum of A53T ?-Synuclein Mutant Mouse Model of Parkinson's Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017953

Elevated levels of α-Syn, p-Tau and p-GSK-3β in striata of α-Syn A53T mutant mice. Striata from wild type non-transgenic mice and A53T α-Syn mutant transgenic mice, 8 months of age, were dissected and homogenized in RIPA buffer, and analyzed by Western blots, as described under “ Materials and Methods ”. After exposure to initial antibodies, blots were stripped and probed for other proteins. The blots show representative gels while the bar graphs are composites summarized from all animals ( n = 4–9). (A) α-Syn and p-α-Syn were expressed relative to β-actin used as a loading control. (B). p-Tau was probed using antibodies specific for pSer202, pSer262 and pSer396/404, and expressed relative to total Tau used as loading control. (C). p-GSK-3β levels were probed using antibodies which recognize phosphorylation at Tyr216, and expressed relative to total GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Asterisks (*) indicate values significantly different from wild-type animals ( P
Figure Legend Snippet: Elevated levels of α-Syn, p-Tau and p-GSK-3β in striata of α-Syn A53T mutant mice. Striata from wild type non-transgenic mice and A53T α-Syn mutant transgenic mice, 8 months of age, were dissected and homogenized in RIPA buffer, and analyzed by Western blots, as described under “ Materials and Methods ”. After exposure to initial antibodies, blots were stripped and probed for other proteins. The blots show representative gels while the bar graphs are composites summarized from all animals ( n = 4–9). (A) α-Syn and p-α-Syn were expressed relative to β-actin used as a loading control. (B). p-Tau was probed using antibodies specific for pSer202, pSer262 and pSer396/404, and expressed relative to total Tau used as loading control. (C). p-GSK-3β levels were probed using antibodies which recognize phosphorylation at Tyr216, and expressed relative to total GSK-3β. (D) PP1, PP2A and PP2B levels were probed using specific antibodies and normalized to β-actin. Asterisks (*) indicate values significantly different from wild-type animals ( P

Techniques Used: Mutagenesis, Mouse Assay, Transgenic Assay, Western Blot

22) Product Images from "Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Can Mediate Degradation of the Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1)"

Article Title: Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Can Mediate Degradation of the Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064145

PCSK9 induces degradation of LRP-1. A ) PCSK9 transfection. HEK293 cells were transfected with PCSK9-V5 or empty control pIRES-V5 vector prior to being lysed in 1x RIPA. LDLR and LRP-1 levels were examined by Western blot in these cells, as well as in HepG2 cells stably expressing PCSK9-shRNA. PCSK9 levels were assessed using mAb-V5 in HEK293 cells and an anti-PCSK9 antibody in HepG2 cells. The levels of LDLR and LRP-1 were estimated relative to β-actin. Data are representative of at least three independent experiments. B ) PCSK9 media swap. Conditioned serum-free media collected from HEK293 cells transfected with PCSK9-V5 or empty control pIRES-V5 vector was collected and applied to naive HEK293 or HepG2 cells. The effect of exogenous PCSK9 on LDLR and LRP-1 was assessed by Western blotting with anti-hLDLR and LRP-1 antibodies respectively. Cell-associated PCSK9 was measured using mAb-V5. The relative intensities of LDLR and LRP-1 were normalized to β-actin using NIH ImageJ software. Data are representative of at least three independent experiments.
Figure Legend Snippet: PCSK9 induces degradation of LRP-1. A ) PCSK9 transfection. HEK293 cells were transfected with PCSK9-V5 or empty control pIRES-V5 vector prior to being lysed in 1x RIPA. LDLR and LRP-1 levels were examined by Western blot in these cells, as well as in HepG2 cells stably expressing PCSK9-shRNA. PCSK9 levels were assessed using mAb-V5 in HEK293 cells and an anti-PCSK9 antibody in HepG2 cells. The levels of LDLR and LRP-1 were estimated relative to β-actin. Data are representative of at least three independent experiments. B ) PCSK9 media swap. Conditioned serum-free media collected from HEK293 cells transfected with PCSK9-V5 or empty control pIRES-V5 vector was collected and applied to naive HEK293 or HepG2 cells. The effect of exogenous PCSK9 on LDLR and LRP-1 was assessed by Western blotting with anti-hLDLR and LRP-1 antibodies respectively. Cell-associated PCSK9 was measured using mAb-V5. The relative intensities of LDLR and LRP-1 were normalized to β-actin using NIH ImageJ software. Data are representative of at least three independent experiments.

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

Independent actions of PCSK9 on the LDLR and LRP-1. A ) PCSK9 acts on LRP-1 independent of the LDLR. CHO-A7 cells were transfected with control empty pIRES vector, PCSK9-V5, LDLR-V5, or both PCSK9-V5 and LDLR-V5. Levels of LRP-1 were measured 24 h after transfection by Western blotting using an anti-LRP-1 antibody. LDLR and PCSK9 were detected using mAb-V5. β-actin levels, as detected by an anti-β-actin antibody, were used to normalize the amounts of LDLR and LRP-1 quantified using NIH ImageJ software. Data are representative of two independent experiments. B ) PCSK9 acts on the LDLR independent of LRP-1. CHO-K1 and CHO 13-5-1 were incubated overnight with conditioned serum-free media collected from HEK293 cells transfected with empty pIRES-V5 vector, PCSK9-V5, or PCSK9 D374Y -V5. The cells were then lysed in 1x RIPA and submitted to Western blotting using the following antibodies: anti-hamster LDLR, anti-LRP-1, mAb-V5 to detect bound PCSK9, and anti-β-actin antibody. Intensities of the LDLR and LRP-1 were normalized to those of β-actin using NIH ImageJ software. Data are representative of three independent experiments.
Figure Legend Snippet: Independent actions of PCSK9 on the LDLR and LRP-1. A ) PCSK9 acts on LRP-1 independent of the LDLR. CHO-A7 cells were transfected with control empty pIRES vector, PCSK9-V5, LDLR-V5, or both PCSK9-V5 and LDLR-V5. Levels of LRP-1 were measured 24 h after transfection by Western blotting using an anti-LRP-1 antibody. LDLR and PCSK9 were detected using mAb-V5. β-actin levels, as detected by an anti-β-actin antibody, were used to normalize the amounts of LDLR and LRP-1 quantified using NIH ImageJ software. Data are representative of two independent experiments. B ) PCSK9 acts on the LDLR independent of LRP-1. CHO-K1 and CHO 13-5-1 were incubated overnight with conditioned serum-free media collected from HEK293 cells transfected with empty pIRES-V5 vector, PCSK9-V5, or PCSK9 D374Y -V5. The cells were then lysed in 1x RIPA and submitted to Western blotting using the following antibodies: anti-hamster LDLR, anti-LRP-1, mAb-V5 to detect bound PCSK9, and anti-β-actin antibody. Intensities of the LDLR and LRP-1 were normalized to those of β-actin using NIH ImageJ software. Data are representative of three independent experiments.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Software, Incubation

PCSK9 acts on the LDLR independent of the receptor's CT and TMD. A ) Generation of chimeric truncated LDLR-V5 constructs. Schematic representation of the LDLR, LDLR lacking its CT (ΔCT), and ΔCT in which the LDLR TMD was swapped with that of ACE2 (ΔCT TMDace2 ) or VLDLR (ΔCT TMDvldlr ). All constructs contained a C-terminal V5-tag. B ) Expression in HEK293 cells. WT and chimeric LDLR constructs were transfected in HEK293 cells. Construct expression was assessed by immunoblotting with mAb-V5. Both mature and immature forms of the LDLR were detected. β-actin was used as a loading control. C) PCSK9 induces LDLR degradation independent of the LDLR's CT and TMD. LDLR, ΔCT, and the ΔCT TMDace2 and ΔCT TMDvldlr chimeric constructs were expressed in HEK293 cells. Twenty-four hours post-transfection, the cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media, which contains both full length PCSK9 and its furin cleaved product at Arg 218 , PCSK9-ΔN 218 [33] . Cells were lysed in 1x RIPA and subjected to Western blot analysis. LDLR and PCSK9 were detected with mAb-V5. β-actin was employed as a loading control. The ability of PCSK9 to induce degradation of the LDLR constructs was quantified using NIH ImageJ software and calculated relative to treatment with pIRES conditioned media. Data are representative of at least three independent experiments. D ) PCSK9 reduces cell surface LDLR levels independent of the receptor's CT and TMD. To assess the ability of PCSK9 added exogenously to HEK293 cells expressing the LDLR or its chimeric constructs, transfected cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media. Subsequently, surface LDLR was quantified by FACS analysis. The values obtained after treatment with PCSK9 are represented graphically relative to treatment with control pIRES. Data are representative of at least three independent experiments. Error bars represent SEM. *, p
Figure Legend Snippet: PCSK9 acts on the LDLR independent of the receptor's CT and TMD. A ) Generation of chimeric truncated LDLR-V5 constructs. Schematic representation of the LDLR, LDLR lacking its CT (ΔCT), and ΔCT in which the LDLR TMD was swapped with that of ACE2 (ΔCT TMDace2 ) or VLDLR (ΔCT TMDvldlr ). All constructs contained a C-terminal V5-tag. B ) Expression in HEK293 cells. WT and chimeric LDLR constructs were transfected in HEK293 cells. Construct expression was assessed by immunoblotting with mAb-V5. Both mature and immature forms of the LDLR were detected. β-actin was used as a loading control. C) PCSK9 induces LDLR degradation independent of the LDLR's CT and TMD. LDLR, ΔCT, and the ΔCT TMDace2 and ΔCT TMDvldlr chimeric constructs were expressed in HEK293 cells. Twenty-four hours post-transfection, the cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media, which contains both full length PCSK9 and its furin cleaved product at Arg 218 , PCSK9-ΔN 218 [33] . Cells were lysed in 1x RIPA and subjected to Western blot analysis. LDLR and PCSK9 were detected with mAb-V5. β-actin was employed as a loading control. The ability of PCSK9 to induce degradation of the LDLR constructs was quantified using NIH ImageJ software and calculated relative to treatment with pIRES conditioned media. Data are representative of at least three independent experiments. D ) PCSK9 reduces cell surface LDLR levels independent of the receptor's CT and TMD. To assess the ability of PCSK9 added exogenously to HEK293 cells expressing the LDLR or its chimeric constructs, transfected cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media. Subsequently, surface LDLR was quantified by FACS analysis. The values obtained after treatment with PCSK9 are represented graphically relative to treatment with control pIRES. Data are representative of at least three independent experiments. Error bars represent SEM. *, p

Techniques Used: Construct, Expressing, Transfection, Plasmid Preparation, Western Blot, Software, FACS

23) Product Images from "Early Cognitive/Social Deficits and Late Motor Phenotype in Conditional Wild-Type TDP-43 Transgenic Mice"

Article Title: Early Cognitive/Social Deficits and Late Motor Phenotype in Conditional Wild-Type TDP-43 Transgenic Mice

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2016.00310

Altered social behavior in TDP-43-WT transgenic (Tg) mice. (A) Experimental design: transgene expression was activated at weaning (postnatal day 28) by removing Dox from water. The behavioral responses of these Tg mice were analyzed at the indicated time points after weaning. (B) Expression of human TAR DNA-binding protein 43 (TDP-43) in Tg mice. Immunoblot of hTDP-43 or total TDP-43 (h+mTDP-43) in cortical RIPA extracts of control (non-Tg) and tTA/WT12 (1, 6 or 12 months off Dox) mice. GAPDH is a loading control. (C) Schematic view of the three-chamber social interaction apparatus, consisting of a black Plexiglas rectangular box with three interconnected chambers. (D) Time spent sniffing the social (S; P21-P28 mouse) or the non–social (NS; black plastic object) stimulus during a 10 min session (test phase) was recorded. 1 month off Dox bigenic mice (tTA/WT12) presented a reduced social interaction time during the session (*** p
Figure Legend Snippet: Altered social behavior in TDP-43-WT transgenic (Tg) mice. (A) Experimental design: transgene expression was activated at weaning (postnatal day 28) by removing Dox from water. The behavioral responses of these Tg mice were analyzed at the indicated time points after weaning. (B) Expression of human TAR DNA-binding protein 43 (TDP-43) in Tg mice. Immunoblot of hTDP-43 or total TDP-43 (h+mTDP-43) in cortical RIPA extracts of control (non-Tg) and tTA/WT12 (1, 6 or 12 months off Dox) mice. GAPDH is a loading control. (C) Schematic view of the three-chamber social interaction apparatus, consisting of a black Plexiglas rectangular box with three interconnected chambers. (D) Time spent sniffing the social (S; P21-P28 mouse) or the non–social (NS; black plastic object) stimulus during a 10 min session (test phase) was recorded. 1 month off Dox bigenic mice (tTA/WT12) presented a reduced social interaction time during the session (*** p

Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Binding Assay

24) Product Images from "c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration"

Article Title: c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration

Journal: FEBS letters

doi: 10.1016/j.febslet.2009.11.009

Macrophage migration requires the c-Abl kinase activity. (A–D) BMDM monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in RIPA buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.
Figure Legend Snippet: Macrophage migration requires the c-Abl kinase activity. (A–D) BMDM monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in RIPA buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.

Techniques Used: Migration, Activity Assay, Transferring, Incubation, Lysis, SDS Page, Western Blot, Immunoprecipitation, Microscopy, Concentration Assay, Expressing, Transfection, Plasmid Preparation, Mutagenesis

25) Product Images from "RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1"

Article Title: RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1

Journal: bioRxiv

doi: 10.1101/555896

RanBP2 promotes the sumoylation and inhibits the ubiquitination of AGO1. (A) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1 , V5-Ubc9 , and His-tagged SUMO2 ( His6-SUMO2 “+”) or control vector ( His6-SUMO2 “-”). 24 h post-transfection cells were lysed in 6 M guanidinium chloride, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, V5-Ubc9, RanBP2 and α-tubulin. (B) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1, and His-Myc-tagged ubiquitin (His-Myc-Ub “+”) or as V5-Ubc9 as a control (His-Myc-Ub “-”). 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in 6 M guanidinium chloride, and the His-Myc-Ub conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His-Myc-Ub conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, RanBP2 and α-tubulin. (C) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 or FH-EYFP. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP and associated proteins were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). They were also analyzed for the immunoprecipitated FH-AGO1/FH-EYFP by immunoblotting against HA, and for co-immunoprecipitated RanBP2. Input lysates were immunoblotted for RanBP2, FH-AGO1 and FH-EYFP (IB: HA) and α-tubulin. (D) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 and either V5-Ubc9 to enhance sumoylation or control plasmid. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). Input lysates were immunoblotted for FH-AGO1 (IB: HA), RanBP2, V5-Ubc9, RanGAP1 and α-tubulin. Note that Ubc9 overexpression, rescues RanGAP1-sumoylation in RanBP2-dE3 cells and decreases the amount of ubiquitinated FH-AGO1.(E) General model for the regulation of IL6 by RanBP2.
Figure Legend Snippet: RanBP2 promotes the sumoylation and inhibits the ubiquitination of AGO1. (A) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1 , V5-Ubc9 , and His-tagged SUMO2 ( His6-SUMO2 “+”) or control vector ( His6-SUMO2 “-”). 24 h post-transfection cells were lysed in 6 M guanidinium chloride, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, V5-Ubc9, RanBP2 and α-tubulin. (B) WT U2OS, and RanBP2-dE3 cells were co-transfected with FH-AGO1, and His-Myc-tagged ubiquitin (His-Myc-Ub “+”) or as V5-Ubc9 as a control (His-Myc-Ub “-”). 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in 6 M guanidinium chloride, and the His-Myc-Ub conjugates were isolated on Nickel beads (“Ni2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His-Myc-Ub conjugates by immunoblotting for His (IB: His). Relative levels of each signal, as analyzed by densitometry, are indicated below each blot. Input lysates were immunoblotted for FH-AGO1, RanBP2 and α-tubulin. (C) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 or FH-EYFP. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP and associated proteins were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). They were also analyzed for the immunoprecipitated FH-AGO1/FH-EYFP by immunoblotting against HA, and for co-immunoprecipitated RanBP2. Input lysates were immunoblotted for RanBP2, FH-AGO1 and FH-EYFP (IB: HA) and α-tubulin. (D) WT U2OS, and RanBP2-dE3 cells were transfected with FH-AGO1 and either V5-Ubc9 to enhance sumoylation or control plasmid. 18 h post-transfection, cells were treated with MG132 (10 µM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and the FH-AGO1/FH-EYFP were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). Input lysates were immunoblotted for FH-AGO1 (IB: HA), RanBP2, V5-Ubc9, RanGAP1 and α-tubulin. Note that Ubc9 overexpression, rescues RanGAP1-sumoylation in RanBP2-dE3 cells and decreases the amount of ubiquitinated FH-AGO1.(E) General model for the regulation of IL6 by RanBP2.

Techniques Used: Transfection, Plasmid Preparation, Isolation, SDS Page, Immunoprecipitation, Over Expression

26) Product Images from "Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells"

Article Title: Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0071316

Specific nucleoporins are degraded in HRV16-infected cells. ( A ) Ohio-HeLa cells were infected without (mock) or with HRV16 (MOI of 1) and cells lysed using RIPA buffer containing protease and phosphatase inhibitors at the time points shown. Cell lysates were subjected to SDS-PAGE on 4–20% gradient gels and Western analysis using the indicated primary antibodies/horseradish peroxidise-conjugated secondary antibodies and enhanced chemiluminescence (Perkin Elmer). The specificity of the antibodies is indicated on the left. Bands corresponding to 3C, 3CD’ and 3CD are indicated on the right. p.i. - post-infection. ( B ) Results for densitometric analysis of FG-Nup protein bands (left) and non-FG-Nups (right) such as those shown in (A), where data were normalised to the corresponding values for tubulin, relative to the corresponding values for the mock sample. Densitometric analyses were performed using Image J; values represent the mean (± SD) from two independent experiments.
Figure Legend Snippet: Specific nucleoporins are degraded in HRV16-infected cells. ( A ) Ohio-HeLa cells were infected without (mock) or with HRV16 (MOI of 1) and cells lysed using RIPA buffer containing protease and phosphatase inhibitors at the time points shown. Cell lysates were subjected to SDS-PAGE on 4–20% gradient gels and Western analysis using the indicated primary antibodies/horseradish peroxidise-conjugated secondary antibodies and enhanced chemiluminescence (Perkin Elmer). The specificity of the antibodies is indicated on the left. Bands corresponding to 3C, 3CD’ and 3CD are indicated on the right. p.i. - post-infection. ( B ) Results for densitometric analysis of FG-Nup protein bands (left) and non-FG-Nups (right) such as those shown in (A), where data were normalised to the corresponding values for tubulin, relative to the corresponding values for the mock sample. Densitometric analyses were performed using Image J; values represent the mean (± SD) from two independent experiments.

Techniques Used: Infection, SDS Page, Western Blot

27) Product Images from "P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance"

Article Title: P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1402845

Clathrin- and caveolae/lipid raft–dependent endocytosis pathways are involved in LL-37 internalization by dTHP-1 cells. HMDMs ( n = 4) ( A ) or dTHP-1 cells ( n = 3) ( B ) were pretreated at 37°C for 1 h with the inhibitors of CME (CLP [10 μM], CLQ [10 μM], or dynasore [20 μM]) or the inhibitors of caveolae/lipid raft–dependent endocytosis (nystatin [10 μg/ml] or filipin [10 μM]). Then the cells were incubated with 10 μg/ml of FAM-labeled LL-37 for an additional hour, and MFI of the cells was analyzed by flow cytometry. ( C ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), CLP (10 μM), or CLQ (25 μM) at 37°C for 1 h and then challenged with LL-37 (10 μg/ml) for an additional hour. After three washes with PBS, the cells were lysed in RIPA buffer. LL-37 in cell lysates was detected by Western blots. The blot is one representative of three independent experiments ( upper panel ). Densitometric analysis of Western blots from three experiments ( lower panel ). ( D ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), dynasore (25 μM), or both inhibitors for 1 h. Subsequently, the cells were challenged with FAM–LL-37 (10 μg/ml), and MFI was analyzed by flow cytometry ( n = 3). ( E ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and CT-B, caveolin-1, or clathrin was visualized using a confocal microscope. ( F ) dTHP-1 cells were treated with TAMRA- or FAM-labeled LL-37 at 37°C for 1 h, and the colocalization of LL-37 and endosome, lysosome, or Golgi was visualized using a confocal microscope. The confocal images are representative of at least three experiments. Images in (E a )–(E c ) and (F a )–(F c ) are enlargements of the boxed areas. Scale bars, 10 μm. * p
Figure Legend Snippet: Clathrin- and caveolae/lipid raft–dependent endocytosis pathways are involved in LL-37 internalization by dTHP-1 cells. HMDMs ( n = 4) ( A ) or dTHP-1 cells ( n = 3) ( B ) were pretreated at 37°C for 1 h with the inhibitors of CME (CLP [10 μM], CLQ [10 μM], or dynasore [20 μM]) or the inhibitors of caveolae/lipid raft–dependent endocytosis (nystatin [10 μg/ml] or filipin [10 μM]). Then the cells were incubated with 10 μg/ml of FAM-labeled LL-37 for an additional hour, and MFI of the cells was analyzed by flow cytometry. ( C ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), CLP (10 μM), or CLQ (25 μM) at 37°C for 1 h and then challenged with LL-37 (10 μg/ml) for an additional hour. After three washes with PBS, the cells were lysed in RIPA buffer. LL-37 in cell lysates was detected by Western blots. The blot is one representative of three independent experiments ( upper panel ). Densitometric analysis of Western blots from three experiments ( lower panel ). ( D ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), dynasore (25 μM), or both inhibitors for 1 h. Subsequently, the cells were challenged with FAM–LL-37 (10 μg/ml), and MFI was analyzed by flow cytometry ( n = 3). ( E ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and CT-B, caveolin-1, or clathrin was visualized using a confocal microscope. ( F ) dTHP-1 cells were treated with TAMRA- or FAM-labeled LL-37 at 37°C for 1 h, and the colocalization of LL-37 and endosome, lysosome, or Golgi was visualized using a confocal microscope. The confocal images are representative of at least three experiments. Images in (E a )–(E c ) and (F a )–(F c ) are enlargements of the boxed areas. Scale bars, 10 μm. * p

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

Macrophages take up neutrophil-derived cathelicidin. Human PMNs were pretreated with CytoB (10 μM) and CaCl 2 (2 mM) for 5 min at 37°C and then treated with LTB 4 (100 nM) for 1 min at 37°C. Subsequently, the culture medium was collected. ( A ) dTHP-1 cells were cultured for 1 h in fresh medium or in the conditioned medium from PMNs that were treated or not with LTB 4 . ( B ) Alternatively, dTHP-1 cells were treated with nystatin (10 μg/ml), dynasore (25 μM), KN-62 (10 μM), or oxATP (100 μM) for 1 h, followed by incubation at 37°C in conditioned medium for 1 h. ( C ) In another experiment, control and P2X 7 R-KD dTHP-1 cells were cultured in conditioned medium for 1 h. Subsequently, dTHP-1 cells were washed with 1× PBS and lysed by RIPA buffer. The LL-37 level in dTHP-1 cells was detected by Western blot. The image is one representative of three independent experiments.
Figure Legend Snippet: Macrophages take up neutrophil-derived cathelicidin. Human PMNs were pretreated with CytoB (10 μM) and CaCl 2 (2 mM) for 5 min at 37°C and then treated with LTB 4 (100 nM) for 1 min at 37°C. Subsequently, the culture medium was collected. ( A ) dTHP-1 cells were cultured for 1 h in fresh medium or in the conditioned medium from PMNs that were treated or not with LTB 4 . ( B ) Alternatively, dTHP-1 cells were treated with nystatin (10 μg/ml), dynasore (25 μM), KN-62 (10 μM), or oxATP (100 μM) for 1 h, followed by incubation at 37°C in conditioned medium for 1 h. ( C ) In another experiment, control and P2X 7 R-KD dTHP-1 cells were cultured in conditioned medium for 1 h. Subsequently, dTHP-1 cells were washed with 1× PBS and lysed by RIPA buffer. The LL-37 level in dTHP-1 cells was detected by Western blot. The image is one representative of three independent experiments.

Techniques Used: Derivative Assay, Cell Culture, Incubation, Western Blot

28) Product Images from "Single amino acid mutation of SR-BI decreases infectivity of hepatitis C virus derived from cell culture in a cell culture model"

Article Title: Single amino acid mutation of SR-BI decreases infectivity of hepatitis C virus derived from cell culture in a cell culture model

Journal: World Journal of Gastroenterology

doi: 10.3748/wjg.v23.i28.5158

Effects of the single amino acid mutant on class B scavenger receptor I mRNA and protein expression. A: Effect of the single amino acid mutant on SR-BI protein expression. Huh7-siSR-BI cells were seeded in 24-well plates, cultured overnight, and transfected with pcDNA3.1 (NC), pcDNA-SR-BI, or pcDNA-SR-BI/S112F. Cells were harvested 72 h after transfection and lysed with RIPA cell lysis buffer; Western blot was performed to analyze the expression of the SR-BI protein. B: Effect of the single amino acid mutant on the SR-BI mRNA level. Huh7-siSR-BI cells were seeded in 24-well plates, cultured overnight, and then transfected with pcDNA3.1 (NC), pcDNA-SR-BI (WT), or pcDNA-SR-BI/S112F (S112F). Cells were harvested 72 h after transfection and RNA was isolated and the level of the SR-BI mRNA was analyzed using qRT-PCR ( a P
Figure Legend Snippet: Effects of the single amino acid mutant on class B scavenger receptor I mRNA and protein expression. A: Effect of the single amino acid mutant on SR-BI protein expression. Huh7-siSR-BI cells were seeded in 24-well plates, cultured overnight, and transfected with pcDNA3.1 (NC), pcDNA-SR-BI, or pcDNA-SR-BI/S112F. Cells were harvested 72 h after transfection and lysed with RIPA cell lysis buffer; Western blot was performed to analyze the expression of the SR-BI protein. B: Effect of the single amino acid mutant on the SR-BI mRNA level. Huh7-siSR-BI cells were seeded in 24-well plates, cultured overnight, and then transfected with pcDNA3.1 (NC), pcDNA-SR-BI (WT), or pcDNA-SR-BI/S112F (S112F). Cells were harvested 72 h after transfection and RNA was isolated and the level of the SR-BI mRNA was analyzed using qRT-PCR ( a P

Techniques Used: Mutagenesis, Expressing, Cell Culture, Transfection, Lysis, Western Blot, Isolation, Quantitative RT-PCR

29) Product Images from "Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM"

Article Title: Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM

Journal: Journal of Virology

doi: 10.1128/JVI.02405-15

Biochemical assessment of the avidity of CDV H-cSLAM interactions. (A to C) Coimmunoprecipitation assays. CDV H FLAG and HA-tagged wt cSLAM, mutant cSLAM, or lion SLAM were coexpressed in Vero cells and subsequently lysed with RIPA buffer 24 h posttransfection.
Figure Legend Snippet: Biochemical assessment of the avidity of CDV H-cSLAM interactions. (A to C) Coimmunoprecipitation assays. CDV H FLAG and HA-tagged wt cSLAM, mutant cSLAM, or lion SLAM were coexpressed in Vero cells and subsequently lysed with RIPA buffer 24 h posttransfection.

Techniques Used: Mutagenesis

30) Product Images from "Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein"

Article Title: Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

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

doi: 10.1073/pnas.0811014106

Ebola GP interacts with tetherin. ( A and B ) 293T cells were transfected with the indicated plasmids, and RIPA lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input
Figure Legend Snippet: Ebola GP interacts with tetherin. ( A and B ) 293T cells were transfected with the indicated plasmids, and RIPA lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input

Techniques Used: Transfection, Immunoprecipitation, Western Blot

31) Product Images from "Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons"

Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

Journal: bioRxiv

doi: 10.1101/2020.01.07.897553

Sp110 −/− mice are not susceptible to M. tuberculosis infections. ( A ), BMMs were treated with 10U/ml of IFNγ for 24 hours and cells were lysed with RIPA buffer. 5μg of total protein was loaded on each lane, and immunoblot was performed with respective antibodies as shown. Molecular weight standards are shown on the left of each blot in kDa. Individual membranes were imaged separately. Three independent lines of Sp100 −/− mice were analyzed (denoted lines 61, 65, and 71). ( B-D ), Lung of mice infected with M. tuberculosis were stained with hematoxylin and eosin (H E) for histology ( B ), measured for CFU at 25 days post-infection (Mann-Whitney test) ( C ) or, monitored for survival ( D ). All except B6 mice were bred in-house, and combined results from the three independent Sp110 −/− lines are shown. Representative of 2 experiments ( B , D ); combined results of 3 infections ( C ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005.
Figure Legend Snippet: Sp110 −/− mice are not susceptible to M. tuberculosis infections. ( A ), BMMs were treated with 10U/ml of IFNγ for 24 hours and cells were lysed with RIPA buffer. 5μg of total protein was loaded on each lane, and immunoblot was performed with respective antibodies as shown. Molecular weight standards are shown on the left of each blot in kDa. Individual membranes were imaged separately. Three independent lines of Sp100 −/− mice were analyzed (denoted lines 61, 65, and 71). ( B-D ), Lung of mice infected with M. tuberculosis were stained with hematoxylin and eosin (H E) for histology ( B ), measured for CFU at 25 days post-infection (Mann-Whitney test) ( C ) or, monitored for survival ( D ). All except B6 mice were bred in-house, and combined results from the three independent Sp110 −/− lines are shown. Representative of 2 experiments ( B , D ); combined results of 3 infections ( C ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005.

Techniques Used: Mouse Assay, Molecular Weight, Infection, Staining, MANN-WHITNEY

32) Product Images from "Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿"

Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿

Journal:

doi: 10.1128/JVI.00913-08

Analysis of gE and gI interaction and glycosylation in melanoma cells. (A) Coimmunoprecipitation of gE and gI in infected cells. Melanoma cells were inoculated with the rOka-ΔCys mutant or rOka control, and lysates were collected in RIPA buffer.
Figure Legend Snippet: Analysis of gE and gI interaction and glycosylation in melanoma cells. (A) Coimmunoprecipitation of gE and gI in infected cells. Melanoma cells were inoculated with the rOka-ΔCys mutant or rOka control, and lysates were collected in RIPA buffer.

Techniques Used: Infection, Mutagenesis

33) Product Images from "Atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 controls the core epithelial-to-mesenchymal transition-inducing transcription factors"

Article Title: Atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 controls the core epithelial-to-mesenchymal transition-inducing transcription factors

Journal: Oncotarget

doi:

Functional domains of Fbxo45 for ubiquitination of Zeb2 A. Lysates from 293T cells transfected Flag-Zeb2 with or without HA-Fbxo45 were immunoprecipitated (IP) with anti-Flag M2 antibody, and then immunoblotted. Total lysates were also used as Input for immunoblotting analysis. B. Bacterially expressed GST or GST–Fbxo45, -Fbxo45ΔF-box, -Fbxo45Δmid, or -Fbxo45ΔSPRY fusion proteins and Glutathione-Sepharose beads were incubated with lysates of HeLa cells transfected with Zeb2, Snai1 or Snai2, respectively. The proteins associated with GST–tagged Fbxo45 forms, bound on the Glutathione-Sepharose beads were washed five times with the RIPA buffer before immunoblotting. C. Lysates from 293T cells transfected Flag-Zeb2 with HA-Fbxo45, -Fbxo45Δmid, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were immunoprecipitated with anti-Flag or anti-HA antibodies, and immunoprecipitates were resolved by SDS-PAGE for Western-blot analysis. D. U2OS cells transfected with Flag-Zeb2 and HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were stained using the primary antibodies of anti-Flag M2 or anti-HA, and the second antibodies of Alexa Fluor 568 anti-mouse or Alexa Fluor 488 anti-Rabbit, respectively. Scale: 25μm. E-I. Zeb2 ubiquitination assays by using IP expriments under different conditions: Flag-Zeb2 with or without HA-Ub (E); Flag-Zeb2 with HA-Ub WT, K48R or K63R (F); Flag-Zeb2 and HA-Ub K48-only with or without myc-Fbox45 (G); Flag-Zeb2 and HA-Ub K48-only with siRNA control, siRNAs for Fbxo45 or siRNAs for Pam (H); Flag-Zeb2 and HA-Ub with HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY (I).
Figure Legend Snippet: Functional domains of Fbxo45 for ubiquitination of Zeb2 A. Lysates from 293T cells transfected Flag-Zeb2 with or without HA-Fbxo45 were immunoprecipitated (IP) with anti-Flag M2 antibody, and then immunoblotted. Total lysates were also used as Input for immunoblotting analysis. B. Bacterially expressed GST or GST–Fbxo45, -Fbxo45ΔF-box, -Fbxo45Δmid, or -Fbxo45ΔSPRY fusion proteins and Glutathione-Sepharose beads were incubated with lysates of HeLa cells transfected with Zeb2, Snai1 or Snai2, respectively. The proteins associated with GST–tagged Fbxo45 forms, bound on the Glutathione-Sepharose beads were washed five times with the RIPA buffer before immunoblotting. C. Lysates from 293T cells transfected Flag-Zeb2 with HA-Fbxo45, -Fbxo45Δmid, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were immunoprecipitated with anti-Flag or anti-HA antibodies, and immunoprecipitates were resolved by SDS-PAGE for Western-blot analysis. D. U2OS cells transfected with Flag-Zeb2 and HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were stained using the primary antibodies of anti-Flag M2 or anti-HA, and the second antibodies of Alexa Fluor 568 anti-mouse or Alexa Fluor 488 anti-Rabbit, respectively. Scale: 25μm. E-I. Zeb2 ubiquitination assays by using IP expriments under different conditions: Flag-Zeb2 with or without HA-Ub (E); Flag-Zeb2 with HA-Ub WT, K48R or K63R (F); Flag-Zeb2 and HA-Ub K48-only with or without myc-Fbox45 (G); Flag-Zeb2 and HA-Ub K48-only with siRNA control, siRNAs for Fbxo45 or siRNAs for Pam (H); Flag-Zeb2 and HA-Ub with HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY (I).

Techniques Used: Functional Assay, Transfection, Immunoprecipitation, Incubation, SDS Page, Western Blot, Staining

34) Product Images from "Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase"

Article Title: Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0225977

Mapping of protease cleavage sites by mass spectrometry. A) Whole cell lysates of Kasumi-1/ctrl and Kasumi-1/shRE cells were prepared in RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche) on day twelve after transduction. Proteins were separated by SDS PAGE, stained with Coomassie blue (n = 3) and subjected to LC-MS analysis. B) The identified peptides were categorized as tryptic peptides, resulting from trypsin digestion during the MS sample preparation procedure, or non-tryptic peptides, resulting from proteolytic cleavage during cell lysis. C) IceLogo showing amino acids at positions P4-P4´, which are enriched or depleted in peptides from Kasumi-1/shRE compared to Kasumi-1/ctrl with p
Figure Legend Snippet: Mapping of protease cleavage sites by mass spectrometry. A) Whole cell lysates of Kasumi-1/ctrl and Kasumi-1/shRE cells were prepared in RIPA buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche) on day twelve after transduction. Proteins were separated by SDS PAGE, stained with Coomassie blue (n = 3) and subjected to LC-MS analysis. B) The identified peptides were categorized as tryptic peptides, resulting from trypsin digestion during the MS sample preparation procedure, or non-tryptic peptides, resulting from proteolytic cleavage during cell lysis. C) IceLogo showing amino acids at positions P4-P4´, which are enriched or depleted in peptides from Kasumi-1/shRE compared to Kasumi-1/ctrl with p

Techniques Used: Mass Spectrometry, Protease Inhibitor, Transduction, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Sample Prep, Lysis

35) Product Images from "The Development and Use of Clickable Activity Based Protein Profiling Agents for Protein Arginine Deiminase 4"

Article Title: The Development and Use of Clickable Activity Based Protein Profiling Agents for Protein Arginine Deiminase 4

Journal: ACS chemical biology

doi: 10.1021/cb1003515

Comparison of labeling methods. (A) Labeling of MCF-7 whole cell extracts with BCA. Extracts, prepared from estrogen stimulated MCF7 cells, were incubated with BCA (1 μM) for 30 min at 37 °C. The labeled proteins were visualized by western blotting using streptavidin conjugated HRP. (B) Post inactivation coupling in live MCF-7 cells. Live estrogen stimulated MCF-7 cells were treated with Cl-amidine-YNE (100 μM) for 60 min. Cells were lysed in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) on ice for 10 min. The samples were denatured at 95 °C for 10 min prior to the addition of the TEV-biotin-azide reporter tag (20 μM). The ‘click’ reaction was then initiated by the addition of TCEP (2.5 mM), ligand (0.119 mM final), and CuSO 4 (5 mM final). The labeled proteins were visualized by western blotting using streptavidin conjugated HRP.
Figure Legend Snippet: Comparison of labeling methods. (A) Labeling of MCF-7 whole cell extracts with BCA. Extracts, prepared from estrogen stimulated MCF7 cells, were incubated with BCA (1 μM) for 30 min at 37 °C. The labeled proteins were visualized by western blotting using streptavidin conjugated HRP. (B) Post inactivation coupling in live MCF-7 cells. Live estrogen stimulated MCF-7 cells were treated with Cl-amidine-YNE (100 μM) for 60 min. Cells were lysed in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) on ice for 10 min. The samples were denatured at 95 °C for 10 min prior to the addition of the TEV-biotin-azide reporter tag (20 μM). The ‘click’ reaction was then initiated by the addition of TCEP (2.5 mM), ligand (0.119 mM final), and CuSO 4 (5 mM final). The labeled proteins were visualized by western blotting using streptavidin conjugated HRP.

Techniques Used: Labeling, BIA-KA, Incubation, Western Blot

36) Product Images from "Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome"

Article Title: Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0180892

SCAMP5 facilitates the secretion of α-synuclein through exosome pathway. (A) Flowchart showing the exosome extraction procedure. (B) Quality control of exosome extraction. Cell lysates(CL), Cell medium(CM), exosome(EXO), Flow through(FT), and RIPA insoluable pellets of EGFP-α-synuclein stable overexpressed SH-SY5Y cells were collected according to the flowchart in 6A. The difference in apparent molecular weight was mainly due to different buffer between samples. Immunoblotting of exosome marker CD63 is conducted using native lysis buffer and native PAGE. Other proteins such as α-synuclein and ER marker Calnexin were examined with SDS-PAGE. (C) Electron microscopy images of exosomes isolated from the cell medium of SH-SY5Y cells stably expressing EGFP-α-synuclein. (D) SCAMP5 is abundant in exosomes, and it increases α-synuclein secretion via exosome. SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with Flag-GFP or Flag-SCAMP5, and harvested 48 hour later. All the samples were processed identically. The loading/total volumes of CL, CM, EXO, and FT were 2/1,200μL, 30/6,000μL, 30/200μL, 30/6,000μL respectively. (E) Quantification of secreted EGFP-α-synuclein in exosome of SH-SY5Y cells overexpressed with SCAMP5 or control in three independent experiments. (mean ±S.E.M.; n = 3; *p
Figure Legend Snippet: SCAMP5 facilitates the secretion of α-synuclein through exosome pathway. (A) Flowchart showing the exosome extraction procedure. (B) Quality control of exosome extraction. Cell lysates(CL), Cell medium(CM), exosome(EXO), Flow through(FT), and RIPA insoluable pellets of EGFP-α-synuclein stable overexpressed SH-SY5Y cells were collected according to the flowchart in 6A. The difference in apparent molecular weight was mainly due to different buffer between samples. Immunoblotting of exosome marker CD63 is conducted using native lysis buffer and native PAGE. Other proteins such as α-synuclein and ER marker Calnexin were examined with SDS-PAGE. (C) Electron microscopy images of exosomes isolated from the cell medium of SH-SY5Y cells stably expressing EGFP-α-synuclein. (D) SCAMP5 is abundant in exosomes, and it increases α-synuclein secretion via exosome. SH-SY5Y cells stably expressing EGFP-α-synuclein were transfected with Flag-GFP or Flag-SCAMP5, and harvested 48 hour later. All the samples were processed identically. The loading/total volumes of CL, CM, EXO, and FT were 2/1,200μL, 30/6,000μL, 30/200μL, 30/6,000μL respectively. (E) Quantification of secreted EGFP-α-synuclein in exosome of SH-SY5Y cells overexpressed with SCAMP5 or control in three independent experiments. (mean ±S.E.M.; n = 3; *p

Techniques Used: Flow Cytometry, Molecular Weight, Marker, Lysis, Clear Native PAGE, SDS Page, Electron Microscopy, Isolation, Stable Transfection, Expressing, Transfection

37) Product Images from "Functional small RNAs are generated from select miRNA hairpin loops in flies and mammals"

Article Title: Functional small RNAs are generated from select miRNA hairpin loops in flies and mammals

Journal: Genes & Development

doi: 10.1101/gad.211698.112

Loading of human miRNA loops to human Ago2. ( A , B ) The indicated miRNAs were overexpressed under the control of the CMV promoter together with myc-tagged human Ago2 in HeLa cells. The myc-Ago2 complex was purified in RIPA buffer. The indicated miRNA loops
Figure Legend Snippet: Loading of human miRNA loops to human Ago2. ( A , B ) The indicated miRNAs were overexpressed under the control of the CMV promoter together with myc-tagged human Ago2 in HeLa cells. The myc-Ago2 complex was purified in RIPA buffer. The indicated miRNA loops

Techniques Used: Purification

38) Product Images from "High-density lipoproteins suppress Aβ-induced PBMC adhesion to human endothelial cells in bioengineered vessels and in monoculture"

Article Title: High-density lipoproteins suppress Aβ-induced PBMC adhesion to human endothelial cells in bioengineered vessels and in monoculture

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-017-0201-0

HDL reduces Aβ association, binding and uptake to hCMEC/D3 whereas blocking Aβ binding or uptake reduces PBMC adhesion to hCMEC/D3. a-d hCMEC/D3 were pre-treated with 100 μg/mL of HDL and 0.1 μM a,c Aβ40 or b,d Aβ42 monomers as described in Fig. 2 at either 37 °C (association a,b ) or at 4 °C (binding c,d ). Cells were lysed in RIPA buffer and Aβ were measured using commercial ELISA. e hCMEC/D3 were pre-treated with HDL (1 mg/mL) for 2 h before stimulating with 1 mM of fluorescently labelled Aβ40 or Aβ42 monomers. Scale bar represents 10 μm. f-n hCMEC/D3 were pre-treated with ( f-h ) RAGE blocking antibody, i-k RAP or l-n heparin or heparinase III 60 min before stimulation with Aβ40 or Aβ42 monomers or TNF-α for 3 h. PBMC adhesion assays were conducted as described in Fig. 2 . Graphs represent means ± SD relative to vehicle treated cells for at least 3 independent trials. * p
Figure Legend Snippet: HDL reduces Aβ association, binding and uptake to hCMEC/D3 whereas blocking Aβ binding or uptake reduces PBMC adhesion to hCMEC/D3. a-d hCMEC/D3 were pre-treated with 100 μg/mL of HDL and 0.1 μM a,c Aβ40 or b,d Aβ42 monomers as described in Fig. 2 at either 37 °C (association a,b ) or at 4 °C (binding c,d ). Cells were lysed in RIPA buffer and Aβ were measured using commercial ELISA. e hCMEC/D3 were pre-treated with HDL (1 mg/mL) for 2 h before stimulating with 1 mM of fluorescently labelled Aβ40 or Aβ42 monomers. Scale bar represents 10 μm. f-n hCMEC/D3 were pre-treated with ( f-h ) RAGE blocking antibody, i-k RAP or l-n heparin or heparinase III 60 min before stimulation with Aβ40 or Aβ42 monomers or TNF-α for 3 h. PBMC adhesion assays were conducted as described in Fig. 2 . Graphs represent means ± SD relative to vehicle treated cells for at least 3 independent trials. * p

Techniques Used: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay

39) Product Images from "Activity-dependent ubiquitination of the AMPA receptor subunit GluA2"

Article Title: Activity-dependent ubiquitination of the AMPA receptor subunit GluA2

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.5944-10.2011

Neuronal activity specifically promotes GluA2 ubiquitination. A) Cortical neurons were incubated in ACSF before bicuculline was added for 15min. Cells were lysed in RIPA or 1% SDS as described under Materials and Methods. AMPARs were immunoprecipitated using non-immune IgG, GluA1 or GluA2 specific antibodies, and immunoblotted for ubiquitin, GluA1, GluA2 and actin. B) Cortical neurons were incubated in ACSF before bicuculline was added for the indicated time. Cells were lysed, and GluA2 was immunoprecipitated using an anti-GluA2 antibody, and immunoblotted for ubiquitin. C) Cortical neurons were incubated in the absence (−) or presence (+) of bicuculline for 30 min at 37°C in ACSF. Bicuculline was washed and neurons in fresh ACSF were returned to 37°C for recovery periods of 15 or 30 min. Cells were lysed and GluA2 was immunoprecipitated using an anti-GluA2 antibody. Samples were subjected to SDS-PAGE followed by immunoblotted with the indicated antibodies.
Figure Legend Snippet: Neuronal activity specifically promotes GluA2 ubiquitination. A) Cortical neurons were incubated in ACSF before bicuculline was added for 15min. Cells were lysed in RIPA or 1% SDS as described under Materials and Methods. AMPARs were immunoprecipitated using non-immune IgG, GluA1 or GluA2 specific antibodies, and immunoblotted for ubiquitin, GluA1, GluA2 and actin. B) Cortical neurons were incubated in ACSF before bicuculline was added for the indicated time. Cells were lysed, and GluA2 was immunoprecipitated using an anti-GluA2 antibody, and immunoblotted for ubiquitin. C) Cortical neurons were incubated in the absence (−) or presence (+) of bicuculline for 30 min at 37°C in ACSF. Bicuculline was washed and neurons in fresh ACSF were returned to 37°C for recovery periods of 15 or 30 min. Cells were lysed and GluA2 was immunoprecipitated using an anti-GluA2 antibody. Samples were subjected to SDS-PAGE followed by immunoblotted with the indicated antibodies.

Techniques Used: Activity Assay, Incubation, Immunoprecipitation, SDS Page

Related Articles

Transfection:

Article Title: Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3
Article Snippet: .. Immunoprecipitation (IP) and Western blotting 293T cells were transiently transfected, harvested 48 hours later, and then lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 50 mM Tris pH 8, 0.1% SDS) containing protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). .. Cell supernatants were incubated with primary anti-tag mAb or polyclonal anti-H3 Ab (0.5–2 ug/ml) centrifuged at 4°C, and then incubated with protein A-Sepharose/protein G PLUS-agarose (Santa Cruz Biotechnology) at 4°C with rotation for 1 hour.

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins
Article Snippet: .. After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). .. Nuclear extracts were prepared to use in the experiment comparing the difference between wild-type SUV39H2 and mutant SUV39H2 (K392A and K392R).

Cell Attachment Assay:

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins
Article Snippet: .. After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). .. Nuclear extracts were prepared to use in the experiment comparing the difference between wild-type SUV39H2 and mutant SUV39H2 (K392A and K392R).

Protease Inhibitor:

Article Title: Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes
Article Snippet: .. Western blot analysis of APP For the analysis of intracellular APP, the cells were washed in PBS and lysed in RIPA-buffer (50 mM HEPES, 150 mM NaCl, 1%(v/v) Igepal, 0.5%(w/v) Na-DOC, 0.1% SDS and 1 tablet Complete Mini protease inhibitor cocktail (Roche, Germany)) on ice. .. After centrifugation the cell lysates were adjusted to equal protein concentrations and boiled for 5 min with the appropriate amount of fourfold concentrated sample buffer to yield a final concentration of 62.5 mM Tris/HCL pH 6.8, 2%(w/v) SDS, 10%(v/v) Glycerol; 100 mM DTT and 0.005%(w/v) bromophenolblue.

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins
Article Snippet: .. After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). .. Nuclear extracts were prepared to use in the experiment comparing the difference between wild-type SUV39H2 and mutant SUV39H2 (K392A and K392R).

Quantitative RT-PCR:

Article Title: Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma
Article Snippet: .. Western blot and RT-qPCR analyses For western blot analysis, whole cell lysates were prepared using RIPA buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). .. Equal amounts of lysate were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA).

Electrophoresis:

Article Title: α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis
Article Snippet: .. Cell extracts were prepared with RIPA buffer (iNtRON, IBS-BR002), resolved on SDS-acrylamide gels by electrophoresis, and transferred to polyvinylidene difluoride membranes (Roche 03010040001). .. The blots were incubated with either 5% non-fat milk (Becton-Dickinson, 232100) or 5% bovine serum albumin (BSA, Affymetrix, 10857) in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 1-3 h. After washing three times with TBST, the blots were reacted with primary antibody at 4 °C overnight followed by secondary antibody at room temperature for 2-4 h, in which primary antibody was diluted in 3-5% BSA and secondary antibody in 5% non-fat milk.

Immunoprecipitation:

Article Title: Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3
Article Snippet: .. Immunoprecipitation (IP) and Western blotting 293T cells were transiently transfected, harvested 48 hours later, and then lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 50 mM Tris pH 8, 0.1% SDS) containing protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). .. Cell supernatants were incubated with primary anti-tag mAb or polyclonal anti-H3 Ab (0.5–2 ug/ml) centrifuged at 4°C, and then incubated with protein A-Sepharose/protein G PLUS-agarose (Santa Cruz Biotechnology) at 4°C with rotation for 1 hour.

Incubation:

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins
Article Snippet: .. After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). .. Nuclear extracts were prepared to use in the experiment comparing the difference between wild-type SUV39H2 and mutant SUV39H2 (K392A and K392R).

Expressing:

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins
Article Snippet: .. After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). .. Nuclear extracts were prepared to use in the experiment comparing the difference between wild-type SUV39H2 and mutant SUV39H2 (K392A and K392R).

Western Blot:

Article Title: Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma
Article Snippet: .. Western blot and RT-qPCR analyses For western blot analysis, whole cell lysates were prepared using RIPA buffer containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). .. Equal amounts of lysate were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA).

Article Title: Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3
Article Snippet: .. Immunoprecipitation (IP) and Western blotting 293T cells were transiently transfected, harvested 48 hours later, and then lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 50 mM Tris pH 8, 0.1% SDS) containing protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). .. Cell supernatants were incubated with primary anti-tag mAb or polyclonal anti-H3 Ab (0.5–2 ug/ml) centrifuged at 4°C, and then incubated with protein A-Sepharose/protein G PLUS-agarose (Santa Cruz Biotechnology) at 4°C with rotation for 1 hour.

Article Title: Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes
Article Snippet: .. Western blot analysis of APP For the analysis of intracellular APP, the cells were washed in PBS and lysed in RIPA-buffer (50 mM HEPES, 150 mM NaCl, 1%(v/v) Igepal, 0.5%(w/v) Na-DOC, 0.1% SDS and 1 tablet Complete Mini protease inhibitor cocktail (Roche, Germany)) on ice. .. After centrifugation the cell lysates were adjusted to equal protein concentrations and boiled for 5 min with the appropriate amount of fourfold concentrated sample buffer to yield a final concentration of 62.5 mM Tris/HCL pH 6.8, 2%(w/v) SDS, 10%(v/v) Glycerol; 100 mM DTT and 0.005%(w/v) bromophenolblue.

Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells
Article Snippet: .. Western blot analysis Cells and exosomes were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline) with complete protease inhibitors (Roche Diagnostics). .. Protein concentration was determined using the Qubit® Protein Assay Kit (Invitrogen).

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    Roche ripa doc buffer
    Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in <t>RIPA-DOC</t> buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p
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    Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in RIPA-DOC buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p

    Journal: Frontiers in Neurology

    Article Title: Low Dose of Apelin-36 Attenuates ER Stress-Associated Apoptosis in Rats with Ischemic Stroke

    doi: 10.3389/fneur.2017.00556

    Figure Lengend Snippet: Low dose of apelin-36 attenuates cerebral I/R injury-induced caspase-3 activation in rats. (A) The MCA territory of sham treated rats, I/R model rats and I/R model rats treated with apelin-36 (labeled I/R-Apelin) were lysed in RIPA-DOC buffer. Cell lysates were resolved on 12% Tris–Glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleaved caspase-3 was detected by cleaved caspase-3 antibody and β-actin served as an internal control was detected by β-actin antibody. (B) Quantification of cleaved caspase-3 expression. The ratio of cleaved caspase-3 to β-actin was further normalized to the sham treated rats. Values represent mean ± SEM. N = 5, * p

    Article Snippet: Western Blot Analysis Brain tissues were homogenized in the RIPA-DOC buffer supplemented with PMSF and phosphatase inhibitor (Roche).

    Techniques: Activation Assay, Labeling, Polyacrylamide Gel Electrophoresis, Expressing

    Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

    Journal: The Journal of Biological Chemistry

    Article Title: Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1) *

    doi: 10.1074/jbc.M113.541102

    Figure Lengend Snippet: Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

    Article Snippet: To obtain the cell lysates, the cells were washed twice with cold PBS and then lysed in RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitors (Roche Applied Science, complete, Mini, EDTA-free), 10 mm NaF, and 1 mm sodium orthovanadate.

    Techniques: Activation Assay, Expressing, Incubation

    Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

    Journal: PLoS ONE

    Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0138849

    Figure Lengend Snippet: Glucose starvation increases exosome secretion in H9C2 cells. (A-B) Representative electron microscopy images of isolated U and P exosomes collected from 90 ml of conditioned medium from H9C2 cells grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (C) Detection of tetraspanins by western blotting of U and P exosome extracts from 90 ml of culture medium from H9C2 cultured as in (A). All exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same amount of RIPA-proteins were loaded in each lane. Graph shows the densitometric analysis of western blot data (n = 3 for U exosomes and n = 1 for P exosomes). (D) WB of CD81, CD9 and Calnexin for 20 μg of exosomal protein isolated by standard ultracentrifugation protocol or 30% sucrose cushion protocol. We didn’t found Calnexin contamination signal for both protocols. Lys: cell lysate (E) Quantification of acetylcholinesterase (Ac Co) activity of exosomes obtained with Exoquick-TC from equal amounts (20 ml) of conditioned medium from H9C2 cells cultured as in (A) (n = 3). A.U. arbitrary units, * P

    Article Snippet: Western blot analysis Cells and exosomes were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline) with complete protease inhibitors (Roche Diagnostics).

    Techniques: Electron Microscopy, Isolation, Western Blot, Cell Culture, Activity Assay

    Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

    Journal: PLoS ONE

    Article Title: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0138849

    Figure Lengend Snippet: Proteomic analysis of rat neonatal CM-derived U exosomes. (A) Representative electron microscopy images of isolated U exosomes collected from 90 ml of conditioned medium from rat neonatal CM grown for 48 h under glucose-starved (+St) or glucose-replete (-St) conditions. Scale bars, 200 nm. (B) Detection of tetraspanins by western blotting of U exosome extracts from 90 ml of conditioned medium from rat neonatal CM cultured as in (A). All U exosome fraction obtained from both experimental condition were resuspended in equal amount of RIPA buffer and the same volume of RIPA-proteins were loaded in each lane. (C) SDS-PAGE electrophoresis and Coomassie blue staining of U exosomal proteins from conditioned medium from rat neonatal CM cultured. U exosome pellets obtained from 90 ml of cultures media were resuspended in RIPA buffer and 30 μg of U exosomal protein from both experimental conditions (+/- St) were loaded in each lane. CD9 and CD81 WB for the same experiment shows equals tetraspanins signaling in both lanes. (D) Protein-protein interaction network obtained using STRING software in U exosomes from rat neonatal CM conditioned medium (+/-St). The images show the confidence view ( http://string-db.org/ ). Stronger associations are represented by thicker lines. (E) Biological processes common or unique to -St or +St treatment group as analyzed using Gene Ontology String software.

    Article Snippet: Western blot analysis Cells and exosomes were lysed in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline) with complete protease inhibitors (Roche Diagnostics).

    Techniques: Derivative Assay, Electron Microscopy, Isolation, Western Blot, Cell Culture, SDS Page, Electrophoresis, Staining, Software

    Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p

    Journal: Theranostics

    Article Title: α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis

    doi: 10.7150/thno.24385

    Figure Lengend Snippet: Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p

    Article Snippet: Cell extracts were prepared with RIPA buffer (iNtRON, IBS-BR002), resolved on SDS-acrylamide gels by electrophoresis, and transferred to polyvinylidene difluoride membranes (Roche 03010040001).

    Techniques: Expressing, Activity Assay, Electrophoresis, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Luciferase