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Cell Signaling Technology Inc biotinylated proteins
Generation of bio ari flies. A) Schematic representation of the domain structure of Drosophila Ari-1 E3 ligase according to Uniprot (ID: Q94981). Ari-1 belongs to the RING between RING E3 ligase family, and as such, it is characterized by the presence of two RING domains (RING1 or R1 from residue 133 to 183 and RING2 or R2 from residue 291 to 322), which are separated by a conserved sequence called the in-between RING domain (IBR, from residue 203 to 264). bio ari flies overexpress an untagged version of Ari-1 in the photoreceptor cells under the control of the GMR-Gal4 driver. B ) Schematic representation of the ( bio Ub) 6 -BirA precursor ( 25 ). It is composed of six copies of ubiquitin, which had been N-terminus modified with a short biotinylatable peptide (MGLNDIFEAQKIEWHEGSGSG). The biotin holoenzyme synthetase (BirA) is found at the C-terminus. This construct is digested by endogenous deubiquitinating enzymes, allowing the biotinylation of each ubiquitin molecule by BirA in vivo . bio ari and bio Ub flies overexpress this precursor in the photoreceptor cells under the control of the GMR-Gal4 driver. C ) Expression of the <t>biotinylated</t> ubiquitin is similar between bio ari and bio Ub flies. Anti-biotin Western blot performed on whole lysates (inputs), as well as on the fractions coming from biotin pulldowns, where the ubiquitinated material is enriched (elutions), is shown. The abundant pyruvate carboxylase (~130 kDa), which is biotinylated endogenously, is highlighted with an arrowhead. D ) Workflow for the identification of Drosophila Ari-1 substrates. 500 mg of heads from bio ari and bio Ub flies were subjected to biotin pulldowns and mass spectrometry analysis. MS data were analyzed by MaxQuant and Perseus softwares.
Biotinylated Proteins, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release"

Article Title: The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release

Journal: bioRxiv

doi: 10.1101/2020.01.23.916619

Generation of bio ari flies. A) Schematic representation of the domain structure of Drosophila Ari-1 E3 ligase according to Uniprot (ID: Q94981). Ari-1 belongs to the RING between RING E3 ligase family, and as such, it is characterized by the presence of two RING domains (RING1 or R1 from residue 133 to 183 and RING2 or R2 from residue 291 to 322), which are separated by a conserved sequence called the in-between RING domain (IBR, from residue 203 to 264). bio ari flies overexpress an untagged version of Ari-1 in the photoreceptor cells under the control of the GMR-Gal4 driver. B ) Schematic representation of the ( bio Ub) 6 -BirA precursor ( 25 ). It is composed of six copies of ubiquitin, which had been N-terminus modified with a short biotinylatable peptide (MGLNDIFEAQKIEWHEGSGSG). The biotin holoenzyme synthetase (BirA) is found at the C-terminus. This construct is digested by endogenous deubiquitinating enzymes, allowing the biotinylation of each ubiquitin molecule by BirA in vivo . bio ari and bio Ub flies overexpress this precursor in the photoreceptor cells under the control of the GMR-Gal4 driver. C ) Expression of the biotinylated ubiquitin is similar between bio ari and bio Ub flies. Anti-biotin Western blot performed on whole lysates (inputs), as well as on the fractions coming from biotin pulldowns, where the ubiquitinated material is enriched (elutions), is shown. The abundant pyruvate carboxylase (~130 kDa), which is biotinylated endogenously, is highlighted with an arrowhead. D ) Workflow for the identification of Drosophila Ari-1 substrates. 500 mg of heads from bio ari and bio Ub flies were subjected to biotin pulldowns and mass spectrometry analysis. MS data were analyzed by MaxQuant and Perseus softwares.
Figure Legend Snippet: Generation of bio ari flies. A) Schematic representation of the domain structure of Drosophila Ari-1 E3 ligase according to Uniprot (ID: Q94981). Ari-1 belongs to the RING between RING E3 ligase family, and as such, it is characterized by the presence of two RING domains (RING1 or R1 from residue 133 to 183 and RING2 or R2 from residue 291 to 322), which are separated by a conserved sequence called the in-between RING domain (IBR, from residue 203 to 264). bio ari flies overexpress an untagged version of Ari-1 in the photoreceptor cells under the control of the GMR-Gal4 driver. B ) Schematic representation of the ( bio Ub) 6 -BirA precursor ( 25 ). It is composed of six copies of ubiquitin, which had been N-terminus modified with a short biotinylatable peptide (MGLNDIFEAQKIEWHEGSGSG). The biotin holoenzyme synthetase (BirA) is found at the C-terminus. This construct is digested by endogenous deubiquitinating enzymes, allowing the biotinylation of each ubiquitin molecule by BirA in vivo . bio ari and bio Ub flies overexpress this precursor in the photoreceptor cells under the control of the GMR-Gal4 driver. C ) Expression of the biotinylated ubiquitin is similar between bio ari and bio Ub flies. Anti-biotin Western blot performed on whole lysates (inputs), as well as on the fractions coming from biotin pulldowns, where the ubiquitinated material is enriched (elutions), is shown. The abundant pyruvate carboxylase (~130 kDa), which is biotinylated endogenously, is highlighted with an arrowhead. D ) Workflow for the identification of Drosophila Ari-1 substrates. 500 mg of heads from bio ari and bio Ub flies were subjected to biotin pulldowns and mass spectrometry analysis. MS data were analyzed by MaxQuant and Perseus softwares.

Techniques Used: Sequencing, Modification, Construct, In Vivo, Expressing, Western Blot, Mass Spectrometry

2) Product Images from "Tamoxifen Directly Interacts with the Dopamine Transporter"

Article Title: Tamoxifen Directly Interacts with the Dopamine Transporter

Journal: The Journal of Pharmacology and Experimental Therapeutics

doi: 10.1124/jpet.118.248179

Tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner. Rat striatal synaptosomes were incubated for 1 hour with 100 μ M cocaine, 10 μ M tamoxifen, or vehicle prior to biotinylation of surface cysteines with maleimide-PEG 2 -biotin. DAT content in biotinylated fractions was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. * P
Figure Legend Snippet: Tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner. Rat striatal synaptosomes were incubated for 1 hour with 100 μ M cocaine, 10 μ M tamoxifen, or vehicle prior to biotinylation of surface cysteines with maleimide-PEG 2 -biotin. DAT content in biotinylated fractions was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. * P

Techniques Used: Incubation, Western Blot

Tamoxifen does not affect surface expression of the DAT. Rat striatal synaptosomes were incubated for 1 hour with 10 μ M tamoxifen or vehicle prior to biotinylation of surface proteins with sulfo-NHS-biotin. After avidin-biotin pulldown, DAT content in biotinylated fractions and lysates was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. (B) Representative Western blots showing the biotinylated fraction blotted for DAT protein and Na + /K + -ATPase, and the corresponding total lysate. Calculations of the ratio (± S.E.M.) of the optical densities of biotinylated transporter–Na + /K + -ATPase were 1.09 ± 0.2 for vehicle and 0.96 ± 0.1 for tamoxifen (data not shown) ( n = 3). TMX, tamoxifen; Veh, vehicle.
Figure Legend Snippet: Tamoxifen does not affect surface expression of the DAT. Rat striatal synaptosomes were incubated for 1 hour with 10 μ M tamoxifen or vehicle prior to biotinylation of surface proteins with sulfo-NHS-biotin. After avidin-biotin pulldown, DAT content in biotinylated fractions and lysates was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. (B) Representative Western blots showing the biotinylated fraction blotted for DAT protein and Na + /K + -ATPase, and the corresponding total lysate. Calculations of the ratio (± S.E.M.) of the optical densities of biotinylated transporter–Na + /K + -ATPase were 1.09 ± 0.2 for vehicle and 0.96 ± 0.1 for tamoxifen (data not shown) ( n = 3). TMX, tamoxifen; Veh, vehicle.

Techniques Used: Expressing, Incubation, Avidin-Biotin Assay, Western Blot

3) Product Images from "Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA (MitomiR) in the Diabetic Heart"

Article Title: Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA (MitomiR) in the Diabetic Heart

Journal: Circulation. Cardiovascular genetics

doi: 10.1161/CIRCGENETICS.115.001067

Crosslinked immunoprecipitation (CLIP) in cardiac mitochondrial subpopulations and MitomiR-378 RISC constituent interactions with the mitochondrial genome. (A) Western blots of biotinylated RNA from CLIP-Ago2 and CLIP-FXR1 reactions illustrating crosslinked protein/RNA and the associated gel shift from 80 kDa to 95–110 kDa. (B) Western blot analyses of CLIP-Ago2 and CLIP-FXR1 subjected to RNAase I treatment at 1:50 dilution (high RNAase) illustrating interaction between the two proteins in the absence of RNA. (C) CLIP-Ago2 and CLIP-FXR1 associated enrichment analyses of mitomiR-378 analyzed by qRT-PCR in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals. (D) CLIP-Ago2 and CLIP-FXR1 associated enrichment analysis of transcripts for mitochondrial encoded ATP6 mRNA levels as assessed by qRT-PCR analysis in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals.
Figure Legend Snippet: Crosslinked immunoprecipitation (CLIP) in cardiac mitochondrial subpopulations and MitomiR-378 RISC constituent interactions with the mitochondrial genome. (A) Western blots of biotinylated RNA from CLIP-Ago2 and CLIP-FXR1 reactions illustrating crosslinked protein/RNA and the associated gel shift from 80 kDa to 95–110 kDa. (B) Western blot analyses of CLIP-Ago2 and CLIP-FXR1 subjected to RNAase I treatment at 1:50 dilution (high RNAase) illustrating interaction between the two proteins in the absence of RNA. (C) CLIP-Ago2 and CLIP-FXR1 associated enrichment analyses of mitomiR-378 analyzed by qRT-PCR in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals. (D) CLIP-Ago2 and CLIP-FXR1 associated enrichment analysis of transcripts for mitochondrial encoded ATP6 mRNA levels as assessed by qRT-PCR analysis in control and diabetic cardiac mitochondrial subpopulations. Values are presented as means ± SE; n = 2 where each individual sample represents a pool of 5 individual animals.

Techniques Used: Immunoprecipitation, Cross-linking Immunoprecipitation, Western Blot, Electrophoretic Mobility Shift Assay, Quantitative RT-PCR

4) Product Images from "RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins"

Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins

Journal: Biochemical pharmacology

doi: 10.1016/j.bcp.2016.12.014

RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P
Figure Legend Snippet: RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P

Techniques Used: Biotin Switch Assay

Identification of C16, C20 and C159 as sites of S-nitrosylation on RhoA. (A) Recombinant RhoA was treated with or without Cys-NO (100 μM) for 30 min and the S-nitrosylation of RhoA was confirmed using the biotin-switch assay and Western blotting. (B–D) Biotinylated RhoA was analyzed by MS/MS and the spectrum of C16, C20 and C159-carbido-Biotin-BMCC tryptic peptides is shown with a summary of the molecular weights of these peptides (top right).
Figure Legend Snippet: Identification of C16, C20 and C159 as sites of S-nitrosylation on RhoA. (A) Recombinant RhoA was treated with or without Cys-NO (100 μM) for 30 min and the S-nitrosylation of RhoA was confirmed using the biotin-switch assay and Western blotting. (B–D) Biotinylated RhoA was analyzed by MS/MS and the spectrum of C16, C20 and C159-carbido-Biotin-BMCC tryptic peptides is shown with a summary of the molecular weights of these peptides (top right).

Techniques Used: Recombinant, Biotin Switch Assay, Western Blot, Mass Spectrometry

Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P
Figure Legend Snippet: Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P

Techniques Used: Mutagenesis, Transfection, Construct, Biotin Switch Assay, Activity Assay, Activation Assay

5) Product Images from "RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins"

Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins

Journal: Biochemical pharmacology

doi: 10.1016/j.bcp.2016.12.014

RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P
Figure Legend Snippet: RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P

Techniques Used: Biotin Switch Assay

Identification of C16, C20 and C159 as sites of S-nitrosylation on RhoA. (A) Recombinant RhoA was treated with or without Cys-NO (100 μM) for 30 min and the S-nitrosylation of RhoA was confirmed using the biotin-switch assay and Western blotting. (B–D) Biotinylated RhoA was analyzed by MS/MS and the spectrum of C16, C20 and C159-carbido-Biotin-BMCC tryptic peptides is shown with a summary of the molecular weights of these peptides (top right).
Figure Legend Snippet: Identification of C16, C20 and C159 as sites of S-nitrosylation on RhoA. (A) Recombinant RhoA was treated with or without Cys-NO (100 μM) for 30 min and the S-nitrosylation of RhoA was confirmed using the biotin-switch assay and Western blotting. (B–D) Biotinylated RhoA was analyzed by MS/MS and the spectrum of C16, C20 and C159-carbido-Biotin-BMCC tryptic peptides is shown with a summary of the molecular weights of these peptides (top right).

Techniques Used: Recombinant, Biotin Switch Assay, Western Blot, Mass Spectrometry

Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P
Figure Legend Snippet: Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P

Techniques Used: Mutagenesis, Transfection, Construct, Biotin Switch Assay, Activity Assay, Activation Assay

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    Interaction among STAT3, PI3K and MAPK. Total protein was isolated from IL-6 treated human 8226 cells transfected with lentivirus bearing shSTAT3 (A) , shPI3K (B) , shMAPK1 (C) , shMAPK2 (D) , or combined shSTAT3 + shPI3K + shMAPK2 (E) , and shRNA control. The proteins were profiled by Western blot with specific antibodies against STAT3, pSTAT3, PI3K, MAPK, pMAPK, MCL-1 or β-actin. Data represented one of two independent experiments.

    Journal: Experimental Hematology & Oncology

    Article Title: Silence of MCL-1 upstream signaling by shRNA abrogates multiple myeloma growth

    doi: 10.1186/2162-3619-3-27

    Figure Lengend Snippet: Interaction among STAT3, PI3K and MAPK. Total protein was isolated from IL-6 treated human 8226 cells transfected with lentivirus bearing shSTAT3 (A) , shPI3K (B) , shMAPK1 (C) , shMAPK2 (D) , or combined shSTAT3 + shPI3K + shMAPK2 (E) , and shRNA control. The proteins were profiled by Western blot with specific antibodies against STAT3, pSTAT3, PI3K, MAPK, pMAPK, MCL-1 or β-actin. Data represented one of two independent experiments.

    Article Snippet: The membranes were treated with mouse anti-STAT3, rabbit anti-MCL-1, anti-PI3K or anti-MAPK1/2 antibodies, rabbit anti-pSTAT-3, anti-pMAPK1/2 antibodies, and mouse anti-β-actin antibodies (1:1000 dilution) (Cell Signaling Technology, USA).

    Techniques: Isolation, Transfection, shRNA, Western Blot

    IL-6 enhanced MCL-1 expression and MM cell growth. Human U266 and 8226 MM cells were cultured in RPMI and stimulated with IL-6 for 0, 24 or 48 hours. (A) MCL-1 gene expression in the cells was quantified by qRT-PCR. mRNA fold change was presented. (B) MCL-1 protein expression was detected by Western blot with anti-MCL-1 specific antibodies. (C) MCL-1 protein expression in IL-6 treated group was quantified based on β-actin level and in comparison with untreated cells. (D and E) U266 and 8226 MM cell growth after IL-6 treatment was measured by MTT assay. Cell growth was presented as OD values. Data represents three independent experiments. *, P

    Journal: Experimental Hematology & Oncology

    Article Title: Silence of MCL-1 upstream signaling by shRNA abrogates multiple myeloma growth

    doi: 10.1186/2162-3619-3-27

    Figure Lengend Snippet: IL-6 enhanced MCL-1 expression and MM cell growth. Human U266 and 8226 MM cells were cultured in RPMI and stimulated with IL-6 for 0, 24 or 48 hours. (A) MCL-1 gene expression in the cells was quantified by qRT-PCR. mRNA fold change was presented. (B) MCL-1 protein expression was detected by Western blot with anti-MCL-1 specific antibodies. (C) MCL-1 protein expression in IL-6 treated group was quantified based on β-actin level and in comparison with untreated cells. (D and E) U266 and 8226 MM cell growth after IL-6 treatment was measured by MTT assay. Cell growth was presented as OD values. Data represents three independent experiments. *, P

    Article Snippet: The membranes were treated with mouse anti-STAT3, rabbit anti-MCL-1, anti-PI3K or anti-MAPK1/2 antibodies, rabbit anti-pSTAT-3, anti-pMAPK1/2 antibodies, and mouse anti-β-actin antibodies (1:1000 dilution) (Cell Signaling Technology, USA).

    Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, MTT Assay

    Effect of shSTAT3, shPI3K and shMAPK1/2 on MCL-1 gene expression. Total RNA was isolated from human U266 and 8226 cells transfected with lentivirus carrying shSTAT3, PI3K shMAPK1, shMAPK2 or control shRNA in presence of IL-6 (48 hours). (A) mRNA expression of STAT3, PI3K and MAPK in U266 cells were quantified by qRT-PCR. The mRNA levels were presented as fold change in comparison with the control shRNA treatment group. (B) Protein expression of STAT3, PI3K or MAPK in U266 cells was determined by Western blot with specific antibodies against the protein. (C) mRNA levels of STAT3, PI3K and MAPK in 8226 cells were also quantified by qRT-PCR and presented as fold changes compared with the shRNA treatment control group. (D) MCL-1 mRNA expression in 8226 cells was quantified by qRT-PCR. The fold change of MCL-1 mRNA in targeted cells was compared with the control shRNA treatment group. *, P

    Journal: Experimental Hematology & Oncology

    Article Title: Silence of MCL-1 upstream signaling by shRNA abrogates multiple myeloma growth

    doi: 10.1186/2162-3619-3-27

    Figure Lengend Snippet: Effect of shSTAT3, shPI3K and shMAPK1/2 on MCL-1 gene expression. Total RNA was isolated from human U266 and 8226 cells transfected with lentivirus carrying shSTAT3, PI3K shMAPK1, shMAPK2 or control shRNA in presence of IL-6 (48 hours). (A) mRNA expression of STAT3, PI3K and MAPK in U266 cells were quantified by qRT-PCR. The mRNA levels were presented as fold change in comparison with the control shRNA treatment group. (B) Protein expression of STAT3, PI3K or MAPK in U266 cells was determined by Western blot with specific antibodies against the protein. (C) mRNA levels of STAT3, PI3K and MAPK in 8226 cells were also quantified by qRT-PCR and presented as fold changes compared with the shRNA treatment control group. (D) MCL-1 mRNA expression in 8226 cells was quantified by qRT-PCR. The fold change of MCL-1 mRNA in targeted cells was compared with the control shRNA treatment group. *, P

    Article Snippet: The membranes were treated with mouse anti-STAT3, rabbit anti-MCL-1, anti-PI3K or anti-MAPK1/2 antibodies, rabbit anti-pSTAT-3, anti-pMAPK1/2 antibodies, and mouse anti-β-actin antibodies (1:1000 dilution) (Cell Signaling Technology, USA).

    Techniques: Expressing, Isolation, Transfection, shRNA, Quantitative RT-PCR, Western Blot

    TRAIL dissembled the RANKL-induced lipid raft-associated signaling complexes. RAW 264.7 cells incubated in minimal essential medium were stimulated by the RANKL with or without the TRAIL for 4 h in the presence and absence of TRAIL-R siRNA or an anti-TRAIL-R blocking antibody (Ab). Cells were lysed with TNE buffer containing 0.5% Brij-58 and subjected to sucrose density gradient centrifugation to isolate lipid raft fractions. Proteins from equal volumes of representative collected fractions were separated by SDS-PAGE and immunoblotted with anti-TRAF6, anti-RANK, anti-c-Src, anti-DAP-12, and anti-flotillin-1 Abs. The lipid raft fraction and cytosolic fraction are presented as fractions 4 and 12, respectively

    Journal: Cell Death & Disease

    Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

    doi: 10.1038/s41419-019-1353-3

    Figure Lengend Snippet: TRAIL dissembled the RANKL-induced lipid raft-associated signaling complexes. RAW 264.7 cells incubated in minimal essential medium were stimulated by the RANKL with or without the TRAIL for 4 h in the presence and absence of TRAIL-R siRNA or an anti-TRAIL-R blocking antibody (Ab). Cells were lysed with TNE buffer containing 0.5% Brij-58 and subjected to sucrose density gradient centrifugation to isolate lipid raft fractions. Proteins from equal volumes of representative collected fractions were separated by SDS-PAGE and immunoblotted with anti-TRAF6, anti-RANK, anti-c-Src, anti-DAP-12, and anti-flotillin-1 Abs. The lipid raft fraction and cytosolic fraction are presented as fractions 4 and 12, respectively

    Article Snippet: Cells were incubated with an anti-RANK, anti-TRAF6 (Santa Cruz Biotechnology, Dallas, TX), anti-c-Src, or anti-DAP-12 antibody (Cell Signaling Technology, Danvers, MA) for 30 min, followed by washing and incubation with an FITC-conjugated secondary antibody for 30 min. Confocal microscopic scanning was performed with a Zeiss LSM-510 META laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

    Techniques: Incubation, Blocking Assay, Gradient Centrifugation, SDS Page

    TRAIL inhibition of RANKL-induced co-localization of TRAF6, RANK, c-Src, and DAP-12 in lipid rafts in confocal microscopy. RAW 264.7 cells were cultured on coverslips and stimulated as indicated. After fixation, cells were processed for immunofluorescence staining of cholera toxin B subunit for GM-1 (red), and Hoechst 33258 (blue). Signaling molecules in lipid rafts were examined by confocal microscopy. a – d The TRAF6 Ab, RANK Ab, Src Ab, and DAP-12 Ab were stained green, and when colocalized with lipid raft GM-1 (red), exhibited yellow staining (merged). (Scale bar represents 10 μm). e – h The percentages of co-localization of RANK, TRAF6, c-Src, and DAP-12 with lipid raft GM-1 were determined and are shown in the bar chart (measured in at least 80 cells. ** p

    Journal: Cell Death & Disease

    Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

    doi: 10.1038/s41419-019-1353-3

    Figure Lengend Snippet: TRAIL inhibition of RANKL-induced co-localization of TRAF6, RANK, c-Src, and DAP-12 in lipid rafts in confocal microscopy. RAW 264.7 cells were cultured on coverslips and stimulated as indicated. After fixation, cells were processed for immunofluorescence staining of cholera toxin B subunit for GM-1 (red), and Hoechst 33258 (blue). Signaling molecules in lipid rafts were examined by confocal microscopy. a – d The TRAF6 Ab, RANK Ab, Src Ab, and DAP-12 Ab were stained green, and when colocalized with lipid raft GM-1 (red), exhibited yellow staining (merged). (Scale bar represents 10 μm). e – h The percentages of co-localization of RANK, TRAF6, c-Src, and DAP-12 with lipid raft GM-1 were determined and are shown in the bar chart (measured in at least 80 cells. ** p

    Article Snippet: Cells were incubated with an anti-RANK, anti-TRAF6 (Santa Cruz Biotechnology, Dallas, TX), anti-c-Src, or anti-DAP-12 antibody (Cell Signaling Technology, Danvers, MA) for 30 min, followed by washing and incubation with an FITC-conjugated secondary antibody for 30 min. Confocal microscopic scanning was performed with a Zeiss LSM-510 META laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

    Techniques: Inhibition, Confocal Microscopy, Cell Culture, Immunofluorescence, Staining

    Time course analysis of dissociation of the RANKL-induced rafts associated signaling complexes after its formation by TRAIL. RAW 264.7 cells were incubated in α-MEM medium and stimulated with RANKL and M-CSF for 4 days to induce the osteoclast differentiation. Then, the RAW 264.7-derived osteoclasts were stimulated with TRAIL and collected cell lysates at different time points (0 min, 15 mins, 30 mins and 60 mins). Cell lysates of RAW 264.7 collected at indicated time points were subjected to SDS-PAGE electrophoresis and immunoblotted with anti-TRAF6, anti-RANK, anti–c-Src, anti-DAP-12, and anti–Flotillin-1 Abs. Lysates of cells without stimulation were used for comparison

    Journal: Cell Death & Disease

    Article Title: TRAIL inhibits RANK signaling and suppresses osteoclast activation via inhibiting lipid raft assembly and TRAF6 recruitment

    doi: 10.1038/s41419-019-1353-3

    Figure Lengend Snippet: Time course analysis of dissociation of the RANKL-induced rafts associated signaling complexes after its formation by TRAIL. RAW 264.7 cells were incubated in α-MEM medium and stimulated with RANKL and M-CSF for 4 days to induce the osteoclast differentiation. Then, the RAW 264.7-derived osteoclasts were stimulated with TRAIL and collected cell lysates at different time points (0 min, 15 mins, 30 mins and 60 mins). Cell lysates of RAW 264.7 collected at indicated time points were subjected to SDS-PAGE electrophoresis and immunoblotted with anti-TRAF6, anti-RANK, anti–c-Src, anti-DAP-12, and anti–Flotillin-1 Abs. Lysates of cells without stimulation were used for comparison

    Article Snippet: Cells were incubated with an anti-RANK, anti-TRAF6 (Santa Cruz Biotechnology, Dallas, TX), anti-c-Src, or anti-DAP-12 antibody (Cell Signaling Technology, Danvers, MA) for 30 min, followed by washing and incubation with an FITC-conjugated secondary antibody for 30 min. Confocal microscopic scanning was performed with a Zeiss LSM-510 META laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

    Techniques: Incubation, Derivative Assay, SDS Page, Electrophoresis

    Alteration of the insulin receptor signaling pathway in adipocytes cultured with amyloid‐β. ( A ) Insulin receptor substrate‐2 ( IRS2 ) gene expression in differentiated adipocytes incubated for 6 days with 100 or 1,000 pg/mL amyloid‐β40 ( n = 10–11 per condition). ( B ) IRS2 transcription in cultures treated with amyloid‐β42 at 10 or 100 pg/mL for 6 days ( n = 10–11 per condition). Representative immunoblot and quantification of ( C ) total Akt‐1 and ( D ), phosphor‐serine 473 Akt‐1 and ( E ) ratio of phospho/total Akt‐1 in adipocyte cultures grown at 5.5 mM glucose with 10 nM insulin with 100 pg/mL or 1,000 pg/mL amyloid‐β40; n = 3 per condition, log‐transformed data. * P

    Journal: Obesity (Silver Spring, Md.)

    Article Title: Effects of glucose and insulin on secretion of amyloid‐β by human adipose tissue cells

    doi: 10.1002/oby.21494

    Figure Lengend Snippet: Alteration of the insulin receptor signaling pathway in adipocytes cultured with amyloid‐β. ( A ) Insulin receptor substrate‐2 ( IRS2 ) gene expression in differentiated adipocytes incubated for 6 days with 100 or 1,000 pg/mL amyloid‐β40 ( n = 10–11 per condition). ( B ) IRS2 transcription in cultures treated with amyloid‐β42 at 10 or 100 pg/mL for 6 days ( n = 10–11 per condition). Representative immunoblot and quantification of ( C ) total Akt‐1 and ( D ), phosphor‐serine 473 Akt‐1 and ( E ) ratio of phospho/total Akt‐1 in adipocyte cultures grown at 5.5 mM glucose with 10 nM insulin with 100 pg/mL or 1,000 pg/mL amyloid‐β40; n = 3 per condition, log‐transformed data. * P

    Article Snippet: Samples were separated on 10% SDS‐PAGE, transferred to PVDF, probed with rabbit monoclonal Akt‐1 antibody (Cell Signaling), followed by goat anti‐rabbit‐HRP‐conjugated antibody (Bio‐Rad), and bands detected using ECL reagent (Amersham).

    Techniques: Cell Culture, Expressing, Incubation, Transformation Assay

    The nuclear translocation of glycogen synthase kinase-3β (GSK-3β) in hippocampal neurons. The control group was untreated. The lipopolysaccharide (LPS) group was treated with LPS (10 μg/mL). Anti-Toll-like receptor 4 (TLR4) + LPS group was pretreated with TLR4 antibody (10 μg/mL) for 2 hours, and then treated with LPS (10 μg/mL). Hippocampal neurons were stained with Alexa Fluor 488-conjugated anti-GSK-3β antibody (green) and Hoechst 33342 (blue). Scale bar: 10 μm.

    Journal: Neural Regeneration Research

    Article Title: Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

    doi: 10.3969/j.issn.1673-5374.2013.29.006

    Figure Lengend Snippet: The nuclear translocation of glycogen synthase kinase-3β (GSK-3β) in hippocampal neurons. The control group was untreated. The lipopolysaccharide (LPS) group was treated with LPS (10 μg/mL). Anti-Toll-like receptor 4 (TLR4) + LPS group was pretreated with TLR4 antibody (10 μg/mL) for 2 hours, and then treated with LPS (10 μg/mL). Hippocampal neurons were stained with Alexa Fluor 488-conjugated anti-GSK-3β antibody (green) and Hoechst 33342 (blue). Scale bar: 10 μm.

    Article Snippet: The membrane was blocked with 5% skim milk powder for 2 hours and incubated with rabbit anti-AKT polyclonal antibody (1:1 000; Cell Signaling, Boston, USA), rabbit anti-P-AKT Ser473 polyclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat GSK-3β monoclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat P-GSK-3βSer9 monoclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat active Caspase-3 monoclonal antibody (1:1 000; Bioworld, Minneapolis, MN, USA), rabbit anti-rat Bcl-2 monoclonal antibody (1:1 000; Bioworld), rabbit anti-rat Bax monoclonal antibody (1:1 000; Bioworld), and mouse anti-rat β-actin monoclonal antibody (1:1 000; Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Translocation Assay, Staining

    Apoptotic rates of hippocampal neurons following inhibition of the PI3K/AKT-GSK-3β signaling pathway detected using flow cytometry. Data are expressed as mean ± SEM ( n = 3). The statistical significance of differences between groups was determined by one-way analysis of variance followed by Tukey's post hoc multiple comparison tests. a P

    Journal: Neural Regeneration Research

    Article Title: Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

    doi: 10.3969/j.issn.1673-5374.2013.29.006

    Figure Lengend Snippet: Apoptotic rates of hippocampal neurons following inhibition of the PI3K/AKT-GSK-3β signaling pathway detected using flow cytometry. Data are expressed as mean ± SEM ( n = 3). The statistical significance of differences between groups was determined by one-way analysis of variance followed by Tukey's post hoc multiple comparison tests. a P

    Article Snippet: The membrane was blocked with 5% skim milk powder for 2 hours and incubated with rabbit anti-AKT polyclonal antibody (1:1 000; Cell Signaling, Boston, USA), rabbit anti-P-AKT Ser473 polyclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat GSK-3β monoclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat P-GSK-3βSer9 monoclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat active Caspase-3 monoclonal antibody (1:1 000; Bioworld, Minneapolis, MN, USA), rabbit anti-rat Bcl-2 monoclonal antibody (1:1 000; Bioworld), rabbit anti-rat Bax monoclonal antibody (1:1 000; Bioworld), and mouse anti-rat β-actin monoclonal antibody (1:1 000; Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Inhibition, Flow Cytometry, Cytometry

    Protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) phosphorylation levels in hippocampal neurons. (A) Representative western-blots for AKT, phosphorylated AKT Ser473 (P-AKT Ser473 ), GSK-3β and phosphorylated GSK-3β Ser9 (P-GSK-3β Ser9 ) protein from hippocampal neurons. (B, C) Quantification of P-AKT Ser473 and P-GSK-3β Ser9 protein levels normalized to the total expression of AKT and GSK3β. The data are expressed as mean ± SEM ( n = 3). The statistical significance of differences between groups was determined by one-way analysis of variance followed by Tukey's post hoc multiple comparison tests. a P

    Journal: Neural Regeneration Research

    Article Title: Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

    doi: 10.3969/j.issn.1673-5374.2013.29.006

    Figure Lengend Snippet: Protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) phosphorylation levels in hippocampal neurons. (A) Representative western-blots for AKT, phosphorylated AKT Ser473 (P-AKT Ser473 ), GSK-3β and phosphorylated GSK-3β Ser9 (P-GSK-3β Ser9 ) protein from hippocampal neurons. (B, C) Quantification of P-AKT Ser473 and P-GSK-3β Ser9 protein levels normalized to the total expression of AKT and GSK3β. The data are expressed as mean ± SEM ( n = 3). The statistical significance of differences between groups was determined by one-way analysis of variance followed by Tukey's post hoc multiple comparison tests. a P

    Article Snippet: The membrane was blocked with 5% skim milk powder for 2 hours and incubated with rabbit anti-AKT polyclonal antibody (1:1 000; Cell Signaling, Boston, USA), rabbit anti-P-AKT Ser473 polyclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat GSK-3β monoclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat P-GSK-3βSer9 monoclonal antibody (1:1 000; Cell Signaling), rabbit anti-rat active Caspase-3 monoclonal antibody (1:1 000; Bioworld, Minneapolis, MN, USA), rabbit anti-rat Bcl-2 monoclonal antibody (1:1 000; Bioworld), rabbit anti-rat Bax monoclonal antibody (1:1 000; Bioworld), and mouse anti-rat β-actin monoclonal antibody (1:1 000; Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Western Blot, Expressing