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Santa Cruz Biotechnology ribonucleic acid
Ribonucleic Acid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 87 stars, based on 1 article reviews
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ribonucleic acid - by Bioz Stars, 2020-04
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Centrifugation:

Article Title: Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways ▿Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways ▿ †
Article Snippet: Briefly, 50 μg of total RNA was extracted from HeLa and HLF cells and incubated with 5 μg of R1131 polyclonal anti-TMG antibody (generous gift from R. Lührmann) or with 5 μg of normal rabbit IgG (sc-2027; Santa Cruz) in 300 μl of IP buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8.0, 250 mM NaCl, 0.05% Nonidet P-40) overnight at 4°C, with continuous rotation. .. The supernatant (SN) and precipitate (beads) were separated by gentle centrifugation.

Article Title: Noncapped Alphavirus Genomic RNAs and Their Role during Infection
Article Snippet: Extracted total RNA from infected cells or UV cross-linked SINV particles were diluted in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.6],150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and bound to either anti-7me G (System Synaptics) or anti-RPS14 (Santa Cruz) antibodies according to the manufacturer's instructions, respectively. .. After incubation while gently mixing at 4°C for a minimum of 4 h, the lysate was clarified via centrifugation prior to being moved to a fresh tube containing protein G Sepharose beads that had been pretreated with 10 μg of both yeast tRNA and bovine serum albumin.

Quantitative RT-PCR:

Article Title: Noncapped Alphavirus Genomic RNAs and Their Role during Infection
Article Snippet: Extracted total RNA from infected cells or UV cross-linked SINV particles were diluted in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.6],150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and bound to either anti-7me G (System Synaptics) or anti-RPS14 (Santa Cruz) antibodies according to the manufacturer's instructions, respectively. .. The resulting immunoprecipitate was used as the template for cDNA synthesis and qRT-PCR detection as previously described ( , ).

Article Title: Transcription Factor KLF5 Binds a Cyclin E1 Polymorphic Intronic Enhancer to Confer Increased Bladder Cancer Risk
Article Snippet: .. Cells were transfected with 10 nM concentrations of siRNA, and RNA was harvested 48 hours post transfection. qRT-PCR and Western blotting were performed to confirm efficient depletion using the following antibodies: GAPDH (sc-47724, Santa Cruz Biotechnology) and KLF5 (ab24331, Abcam). .. Enhancer capabilities of the CCNE1 intronic region were assessed using the Dual-Luciferase expression assay (Promega) according to manufacturer's instructions.

Real-time Polymerase Chain Reaction:

Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress
Article Snippet: To isolate RNA bound with EF1a protein, 2 μg of anti-EF1a antibody (H-300; Santa Cruz Biotechnology, CA) or rabbit IgG (catalog number sc-2027; Santa Cruz Biotechnology, CA) was first incubated with 100 μl of protein A/G Plus beads (catalog number sc-2003; Santa Cruz Biotechnology, CA) for 1 h at 25°C ( ). .. For quantitative measurements of NRF2 mRNA, the cDNA was used as a template for quantitative PCR (qPCR) using a Bio-Rad CFX96 thermocycler with the primer pair 5′-CAGGTTGCCCACATTCCCAAATCA-3′ and 5′-AGCAATGAAGACTGGGCTCTCGAT-3′ for human NRF2 (NCBI RefSeq accession number ).

Incubation:

Article Title: Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer. Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer
Article Snippet: 2.6 Small interfering RNA and shRNA transfection Knockdown of OTC was carried out by siRNA; all related reagents were purchased from Santa Cruz Biotechnology. .. Briefly, a mixture of siRNA and transfection reagent was incubated with cells for 6 hours, then changed to complete medium for 48 hours of incubation.

Article Title: Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach
Article Snippet: .. Twelve milligrams of total RNA was incubated with 300 µg of anti-TMG (K121) monoclonal antibody (Santa Cruz Biotechnology) in 1X IP buffer (25 mM Tris-HCl at pH 8.0, 200 mM NaCl, and 0.2 mM EDTA) with 0.1 units/µL SUPERase-In RNase inhibitor (Ambion) at 4°C for 2 h. The mixture was then combined with 400 µL of Ultralink Immobilized Protein A/G gel slurry (Pierce) that was preblocked with 1 mg/mL of RNase-free BSA (Ambion) in 1X IP buffer at 4°C for 20 min with gentle agitation. .. The gel slurry was washed five times with 1X Wash buffer (25 mM Tris-HCl at pH 8.0, 300 mM NaCl, and 0.2 mM EDTA) and eluted with 1X SDS Elution buffer (25 mM Tris-HCl at pH 8.0, 200 mM NaCl, 5 mM EDTA, and 1% SDS) at room temperature for 10 min. RNA was isolated by acid phenol/chloroform extraction and ethanol precipitation.

Article Title: (−)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase
Article Snippet: HCAEC were transfected with small interfering RNA [siRNA; sieNOS (sc-36093) or scrambled RNA duplex as a control (sc-37007) from Santa Cruz Biotechnology] using the transfection reagent (sc-29528, Santa Cruz Biotechnology) according to the manufacturer’s instructions. .. Thereafter, cells were electroporated using the siRNA transfection reagent and incubated for 8 h with 60 nmol siRNA based on the manufacturer’s protocol.

Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress
Article Snippet: .. To isolate RNA bound with EF1a protein, 2 μg of anti-EF1a antibody (H-300; Santa Cruz Biotechnology, CA) or rabbit IgG (catalog number sc-2027; Santa Cruz Biotechnology, CA) was first incubated with 100 μl of protein A/G Plus beads (catalog number sc-2003; Santa Cruz Biotechnology, CA) for 1 h at 25°C ( ). .. The unbound antibodies were removed by washing the beads 3 times with NAB buffer, followed by incubation with 500 μg HEK293 cytoplasmic extracts at 4°C for 4 h. The unbound proteins were removed by 5 washes with NAB buffer.

Article Title: A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy
Article Snippet: Then, RNase A (1 μg/ml) was added to supernatants and the samples were incubated for 1 h at 37°C prior to realize an immunoprecipitation with an UPF1 antibody. .. RNA was immunoprecipitated using 4 μg of UPF1 antibody and 4 μg of control rabbit IgG (sc-2027, Santa Cruz).

Article Title: Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways ▿Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways ▿ †
Article Snippet: .. Briefly, 50 μg of total RNA was extracted from HeLa and HLF cells and incubated with 5 μg of R1131 polyclonal anti-TMG antibody (generous gift from R. Lührmann) or with 5 μg of normal rabbit IgG (sc-2027; Santa Cruz) in 300 μl of IP buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8.0, 250 mM NaCl, 0.05% Nonidet P-40) overnight at 4°C, with continuous rotation. .. The RNA-antibody complexes were coupled to 50 μl of 50% slurry of protein A-Sepharose (GE Healthcare), prewashed three times with IP buffer for 2 h at 4°C.

Article Title: Noncapped Alphavirus Genomic RNAs and Their Role during Infection
Article Snippet: Extracted total RNA from infected cells or UV cross-linked SINV particles were diluted in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.6],150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and bound to either anti-7me G (System Synaptics) or anti-RPS14 (Santa Cruz) antibodies according to the manufacturer's instructions, respectively. .. After incubation while gently mixing at 4°C for a minimum of 4 h, the lysate was clarified via centrifugation prior to being moved to a fresh tube containing protein G Sepharose beads that had been pretreated with 10 μg of both yeast tRNA and bovine serum albumin.

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: .. In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz). ..

Infection:

Article Title: Noncapped Alphavirus Genomic RNAs and Their Role during Infection
Article Snippet: .. Extracted total RNA from infected cells or UV cross-linked SINV particles were diluted in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.6],150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and bound to either anti-7me G (System Synaptics) or anti-RPS14 (Santa Cruz) antibodies according to the manufacturer's instructions, respectively. .. After incubation while gently mixing at 4°C for a minimum of 4 h, the lysate was clarified via centrifugation prior to being moved to a fresh tube containing protein G Sepharose beads that had been pretreated with 10 μg of both yeast tRNA and bovine serum albumin.

Expressing:

Article Title: Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque
Article Snippet: Transfection of small interfering RNA into cells The fibroblasts were serum-starved for 24 hours and transfected with 100 pmol small interfering RNA (siRNA) oligonucleotides targeted specifically to HDAC7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by using Lipofectamine 2000 (GIBCO). .. The fibroblasts were then treated with 10 ng/mL TGF-β1 (R & D Systems Inc., Minneapolis, MN, USA) for 24 hours to detect the protein expression of plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen subtypes, smooth muscle α-actin, and HDAC7.

Western Blot:

Article Title: Transcription Factor KLF5 Binds a Cyclin E1 Polymorphic Intronic Enhancer to Confer Increased Bladder Cancer Risk
Article Snippet: .. Cells were transfected with 10 nM concentrations of siRNA, and RNA was harvested 48 hours post transfection. qRT-PCR and Western blotting were performed to confirm efficient depletion using the following antibodies: GAPDH (sc-47724, Santa Cruz Biotechnology) and KLF5 (ab24331, Abcam). .. Enhancer capabilities of the CCNE1 intronic region were assessed using the Dual-Luciferase expression assay (Promega) according to manufacturer's instructions.

RNA Binding Assay:

Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress
Article Snippet: Paragraph title: Immunoprecipitation of the RNA binding protein complex. ... To isolate RNA bound with EF1a protein, 2 μg of anti-EF1a antibody (H-300; Santa Cruz Biotechnology, CA) or rabbit IgG (catalog number sc-2027; Santa Cruz Biotechnology, CA) was first incubated with 100 μl of protein A/G Plus beads (catalog number sc-2003; Santa Cruz Biotechnology, CA) for 1 h at 25°C ( ).

Transfection:

Article Title: Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer. Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer
Article Snippet: .. 2.6 Small interfering RNA and shRNA transfection Knockdown of OTC was carried out by siRNA; all related reagents were purchased from Santa Cruz Biotechnology. .. Briefly, a mixture of siRNA and transfection reagent was incubated with cells for 6 hours, then changed to complete medium for 48 hours of incubation.

Article Title: (−)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase
Article Snippet: .. HCAEC were transfected with small interfering RNA [siRNA; sieNOS (sc-36093) or scrambled RNA duplex as a control (sc-37007) from Santa Cruz Biotechnology] using the transfection reagent (sc-29528, Santa Cruz Biotechnology) according to the manufacturer’s instructions. ..

Article Title: Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis
Article Snippet: Nucleofector transfection kits were from Amaxa (Gaithersbury, MD, USA). .. Goat polyclonal FBXL2 antibody, scrambled RNA and siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque
Article Snippet: .. Transfection of small interfering RNA into cells The fibroblasts were serum-starved for 24 hours and transfected with 100 pmol small interfering RNA (siRNA) oligonucleotides targeted specifically to HDAC7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by using Lipofectamine 2000 (GIBCO). ..

Article Title: Transcription Factor KLF5 Binds a Cyclin E1 Polymorphic Intronic Enhancer to Confer Increased Bladder Cancer Risk
Article Snippet: .. Cells were transfected with 10 nM concentrations of siRNA, and RNA was harvested 48 hours post transfection. qRT-PCR and Western blotting were performed to confirm efficient depletion using the following antibodies: GAPDH (sc-47724, Santa Cruz Biotechnology) and KLF5 (ab24331, Abcam). .. Enhancer capabilities of the CCNE1 intronic region were assessed using the Dual-Luciferase expression assay (Promega) according to manufacturer's instructions.

Nick Translation:

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: DNA probe, made of eleven PCR amplifications ( ) was labeled with digoxigenin using nick translation kit (Boehringer). .. In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz).

Cell Culture:

Article Title: Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque
Article Snippet: Transfection of small interfering RNA into cells The fibroblasts were serum-starved for 24 hours and transfected with 100 pmol small interfering RNA (siRNA) oligonucleotides targeted specifically to HDAC7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by using Lipofectamine 2000 (GIBCO). .. After transfection, cells were plated and cultured for 48 hours in DMEM.

DNA Sequencing:

Article Title: Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis
Article Snippet: Goat polyclonal FBXL2 antibody, scrambled RNA and siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). .. All DNA sequencing was performed by the University of Pittsburgh DNA Core Facility.

Polymerase Chain Reaction:

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: DNA probe, made of eleven PCR amplifications ( ) was labeled with digoxigenin using nick translation kit (Boehringer). .. In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz).

Isolation:

Article Title: Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach
Article Snippet: Twelve milligrams of total RNA was incubated with 300 µg of anti-TMG (K121) monoclonal antibody (Santa Cruz Biotechnology) in 1X IP buffer (25 mM Tris-HCl at pH 8.0, 200 mM NaCl, and 0.2 mM EDTA) with 0.1 units/µL SUPERase-In RNase inhibitor (Ambion) at 4°C for 2 h. The mixture was then combined with 400 µL of Ultralink Immobilized Protein A/G gel slurry (Pierce) that was preblocked with 1 mg/mL of RNase-free BSA (Ambion) in 1X IP buffer at 4°C for 20 min with gentle agitation. .. The gel slurry was washed five times with 1X Wash buffer (25 mM Tris-HCl at pH 8.0, 300 mM NaCl, and 0.2 mM EDTA) and eluted with 1X SDS Elution buffer (25 mM Tris-HCl at pH 8.0, 200 mM NaCl, 5 mM EDTA, and 1% SDS) at room temperature for 10 min. RNA was isolated by acid phenol/chloroform extraction and ethanol precipitation.

Labeling:

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: DNA probe, made of eleven PCR amplifications ( ) was labeled with digoxigenin using nick translation kit (Boehringer). .. In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz).

Small Interfering RNA:

Article Title: Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer. Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer
Article Snippet: .. 2.6 Small interfering RNA and shRNA transfection Knockdown of OTC was carried out by siRNA; all related reagents were purchased from Santa Cruz Biotechnology. .. Briefly, a mixture of siRNA and transfection reagent was incubated with cells for 6 hours, then changed to complete medium for 48 hours of incubation.

Article Title: (−)-Epicatechin-induced recovery of mitochondria from simulated diabetes: Potential role of endothelial nitric oxide synthase
Article Snippet: .. HCAEC were transfected with small interfering RNA [siRNA; sieNOS (sc-36093) or scrambled RNA duplex as a control (sc-37007) from Santa Cruz Biotechnology] using the transfection reagent (sc-29528, Santa Cruz Biotechnology) according to the manufacturer’s instructions. ..

Article Title: Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque
Article Snippet: .. Transfection of small interfering RNA into cells The fibroblasts were serum-starved for 24 hours and transfected with 100 pmol small interfering RNA (siRNA) oligonucleotides targeted specifically to HDAC7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by using Lipofectamine 2000 (GIBCO). ..

FACS:

Article Title: Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis
Article Snippet: FACS kits were purchased from BD Biosciences (San Jose, CA, USA). .. Goat polyclonal FBXL2 antibody, scrambled RNA and siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Microscopy:

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz). .. Three-dimensional images were acquired from stage 6 egg chambers on a Leica SP5 confocal microscope using a 40X objective.

Staining:

Article Title: Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis
Article Snippet: Annexin V staining kits and the complete proteasome inhibitors were from Roche (Madison, WI, USA). .. Goat polyclonal FBXL2 antibody, scrambled RNA and siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: After washes, ovaries were first incubated with glycine 0.1 M–0.1% Tween HCL pH2.2 before washes and DNA staining. .. In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz).

In Situ Hybridization:

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: Paragraph title: In Situ Hybridization ... In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz).

Negative Control:

Article Title: Transcription Factor KLF5 Binds a Cyclin E1 Polymorphic Intronic Enhancer to Confer Increased Bladder Cancer Risk
Article Snippet: Two Silencer Select siRNAs (Life Technologies) against KLF5 or a scrambled negative control were used for RNAi experiments. .. Cells were transfected with 10 nM concentrations of siRNA, and RNA was harvested 48 hours post transfection. qRT-PCR and Western blotting were performed to confirm efficient depletion using the following antibodies: GAPDH (sc-47724, Santa Cruz Biotechnology) and KLF5 (ab24331, Abcam).

shRNA:

Article Title: Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer. Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer
Article Snippet: .. 2.6 Small interfering RNA and shRNA transfection Knockdown of OTC was carried out by siRNA; all related reagents were purchased from Santa Cruz Biotechnology. .. Briefly, a mixture of siRNA and transfection reagent was incubated with cells for 6 hours, then changed to complete medium for 48 hours of incubation.

Radio Immunoprecipitation:

Article Title: Noncapped Alphavirus Genomic RNAs and Their Role during Infection
Article Snippet: .. Extracted total RNA from infected cells or UV cross-linked SINV particles were diluted in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.6],150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and bound to either anti-7me G (System Synaptics) or anti-RPS14 (Santa Cruz) antibodies according to the manufacturer's instructions, respectively. .. After incubation while gently mixing at 4°C for a minimum of 4 h, the lysate was clarified via centrifugation prior to being moved to a fresh tube containing protein G Sepharose beads that had been pretreated with 10 μg of both yeast tRNA and bovine serum albumin.

Ethanol Precipitation:

Article Title: Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach
Article Snippet: Twelve milligrams of total RNA was incubated with 300 µg of anti-TMG (K121) monoclonal antibody (Santa Cruz Biotechnology) in 1X IP buffer (25 mM Tris-HCl at pH 8.0, 200 mM NaCl, and 0.2 mM EDTA) with 0.1 units/µL SUPERase-In RNase inhibitor (Ambion) at 4°C for 2 h. The mixture was then combined with 400 µL of Ultralink Immobilized Protein A/G gel slurry (Pierce) that was preblocked with 1 mg/mL of RNase-free BSA (Ambion) in 1X IP buffer at 4°C for 20 min with gentle agitation. .. The gel slurry was washed five times with 1X Wash buffer (25 mM Tris-HCl at pH 8.0, 300 mM NaCl, and 0.2 mM EDTA) and eluted with 1X SDS Elution buffer (25 mM Tris-HCl at pH 8.0, 200 mM NaCl, 5 mM EDTA, and 1% SDS) at room temperature for 10 min. RNA was isolated by acid phenol/chloroform extraction and ethanol precipitation.

Immunoprecipitation:

Article Title: Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach
Article Snippet: Paragraph title: TMG immunoprecipitation of total RNA ... Twelve milligrams of total RNA was incubated with 300 µg of anti-TMG (K121) monoclonal antibody (Santa Cruz Biotechnology) in 1X IP buffer (25 mM Tris-HCl at pH 8.0, 200 mM NaCl, and 0.2 mM EDTA) with 0.1 units/µL SUPERase-In RNase inhibitor (Ambion) at 4°C for 2 h. The mixture was then combined with 400 µL of Ultralink Immobilized Protein A/G gel slurry (Pierce) that was preblocked with 1 mg/mL of RNase-free BSA (Ambion) in 1X IP buffer at 4°C for 20 min with gentle agitation.

Article Title: G-Quadruplex in the NRF2 mRNA 5′ Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress
Article Snippet: Paragraph title: Immunoprecipitation of the RNA binding protein complex. ... To isolate RNA bound with EF1a protein, 2 μg of anti-EF1a antibody (H-300; Santa Cruz Biotechnology, CA) or rabbit IgG (catalog number sc-2027; Santa Cruz Biotechnology, CA) was first incubated with 100 μl of protein A/G Plus beads (catalog number sc-2003; Santa Cruz Biotechnology, CA) for 1 h at 25°C ( ).

Article Title: A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy
Article Snippet: .. RNA was immunoprecipitated using 4 μg of UPF1 antibody and 4 μg of control rabbit IgG (sc-2027, Santa Cruz). .. Immunoblotting The following antibodies were used: CARM1 (A300–421A, Bethyl Laboratories); GAPDH (MMS-580S, Covance); β-Actin (sc-47778, Santa Cruz Biotechnology); Tubulin (T6199, Sigma-Aldrich); UPF1 (07–1014, Millipore); UPF2 (D3B10, Cell Signaling); UPF3b (sc-48800, Santa Cruz Biotechnology); Magoh (Ab10686, Abcam); eIF4A3 (Ab32485, Abcam); RBM8a (NB100–55326, Novus Biologicals); Casc3 (LS-C100827, Lifespan Biosciences); Asparagine Synthetase (sc365809, Santa Cruz Biotechnology); Arc (sc15325, Santa Cruz Biotechnology); GADD45a (sc-796, Santa Cruz Biotechnology).

Article Title: Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways ▿Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways ▿ †
Article Snippet: Paragraph title: Immunoprecipitation of TMG-capped RNAs. ... Briefly, 50 μg of total RNA was extracted from HeLa and HLF cells and incubated with 5 μg of R1131 polyclonal anti-TMG antibody (generous gift from R. Lührmann) or with 5 μg of normal rabbit IgG (sc-2027; Santa Cruz) in 300 μl of IP buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8.0, 250 mM NaCl, 0.05% Nonidet P-40) overnight at 4°C, with continuous rotation.

Article Title: Noncapped Alphavirus Genomic RNAs and Their Role during Infection
Article Snippet: Paragraph title: Immunoprecipitation. ... Extracted total RNA from infected cells or UV cross-linked SINV particles were diluted in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.6],150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and bound to either anti-7me G (System Synaptics) or anti-RPS14 (Santa Cruz) antibodies according to the manufacturer's instructions, respectively.

Article Title: miR-320a mediates doxorubicin-induced cardiotoxicity by targeting VEGF signal pathway
Article Snippet: .. Co-immunoprecipitation of RNA with anti-Ago2 antibody HUVEC were lysed and then immunoprecipitated with anti-Ago2 antibody (Santa Cruz, CA, USA) by protein G Sepharose beads as previously described [ ]. .. BrdU assay of cell proliferation HUVEC cells were labeled with BrdU according to the manufacturer's protocol (EMD Chemicals, Darmstadt, Germany).

Lysis:

Article Title: A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy
Article Snippet: For RNase A treatment (Qiagen, 19101), the cell pellets were first incubated with lysis buffer. .. RNA was immunoprecipitated using 4 μg of UPF1 antibody and 4 μg of control rabbit IgG (sc-2027, Santa Cruz).

Fluorescence In Situ Hybridization:

Article Title: "Dot COM", a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila
Article Snippet: For DNA/RNA in situ hybridization, RNA staining was followed by treatment with 200 µg/ml RNase A for 2 hrs and ovaries were then transferred to FISH hybridization buffer containing 50% formamide, 4X SSC, 0,1% Tween 20, 0,1 M NaH2PO4. .. In RNA-immuno-FISH, RNA straining was followed by incubation with mouse anti-lamin antibody (ADL67-10, Hybridoma), mouse anti-Armitage antibody or rabbit anti-GFP antibody (sc-8334, Santa Cruz).

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    Santa Cruz Biotechnology tsg 6 sirna
    In vitro effect of <t>TSG-6</t> on caerulein- and LPS-stimulated primary pancreatic acinar cells (PACs). a TSG-6 mRNA expression levels in hAT-MSCs transfected with TSG-6 <t>siRNA</t> (TSG-6 siRNA MSCs), hAT-MSCs transfected with control siRNA (scr siRNA MSCs), and naive hAT-MSCs (MSCs). b , c Caerulein- and LPS-stimulated primary PACs were indirectly co-cultured with each of the three hAT-MSC types for 12 h. b Grp78, CHOP, and caspase-12 mRNA expression levels in primary PACs. c Grp78, CHOP, and caspase-12 protein expression levels. d The expression levels of NF-κB p65 activity were analysed by western blotting. e TUNEL staining in PACs in each group observed at ×200 magnification. TUNEL staining revealed the number of cells undergoing apoptosis or pyroptosis. Results are presented as the mean ± SD obtained from three independent experiments * P
    Tsg 6 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology cd38
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    In vitro effect of TSG-6 on caerulein- and LPS-stimulated primary pancreatic acinar cells (PACs). a TSG-6 mRNA expression levels in hAT-MSCs transfected with TSG-6 siRNA (TSG-6 siRNA MSCs), hAT-MSCs transfected with control siRNA (scr siRNA MSCs), and naive hAT-MSCs (MSCs). b , c Caerulein- and LPS-stimulated primary PACs were indirectly co-cultured with each of the three hAT-MSC types for 12 h. b Grp78, CHOP, and caspase-12 mRNA expression levels in primary PACs. c Grp78, CHOP, and caspase-12 protein expression levels. d The expression levels of NF-κB p65 activity were analysed by western blotting. e TUNEL staining in PACs in each group observed at ×200 magnification. TUNEL staining revealed the number of cells undergoing apoptosis or pyroptosis. Results are presented as the mean ± SD obtained from three independent experiments * P

    Journal: Stem Cell Research & Therapy

    Article Title: TSG-6 secreted by human adipose tissue-derived mesenchymal stem cells ameliorates severe acute pancreatitis via ER stress downregulation in mice

    doi: 10.1186/s13287-018-1009-8

    Figure Lengend Snippet: In vitro effect of TSG-6 on caerulein- and LPS-stimulated primary pancreatic acinar cells (PACs). a TSG-6 mRNA expression levels in hAT-MSCs transfected with TSG-6 siRNA (TSG-6 siRNA MSCs), hAT-MSCs transfected with control siRNA (scr siRNA MSCs), and naive hAT-MSCs (MSCs). b , c Caerulein- and LPS-stimulated primary PACs were indirectly co-cultured with each of the three hAT-MSC types for 12 h. b Grp78, CHOP, and caspase-12 mRNA expression levels in primary PACs. c Grp78, CHOP, and caspase-12 protein expression levels. d The expression levels of NF-κB p65 activity were analysed by western blotting. e TUNEL staining in PACs in each group observed at ×200 magnification. TUNEL staining revealed the number of cells undergoing apoptosis or pyroptosis. Results are presented as the mean ± SD obtained from three independent experiments * P

    Article Snippet: The results showed that treatment with naive hAT-MSCs or hAT-MSCs transduced with scr siRNA may significantly reduce the apoptotic ratio, compared to the case in the positive control group; however, we failed to observe similar effects with hAT-MSCs transduced with TSG-6 siRNA.

    Techniques: In Vitro, Expressing, HAT Assay, Transfection, Cell Culture, Activity Assay, Western Blot, TUNEL Assay, Staining

    Schematic representation visualizing the major findings of the study. The study reveals the presence of distributed nanocores within the mobile, liquid droplet-like phase of SGs. The two RBPs, G3BP1 (green arrows) and IMP1 (red arrows), exhibit markedly different SG–cytosol interconversion dynamics. Within granules, both RBPs exhibit alternating binding in the nanocores and liquid droplet-like diffusion in the mobile phase.

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: Schematic representation visualizing the major findings of the study. The study reveals the presence of distributed nanocores within the mobile, liquid droplet-like phase of SGs. The two RBPs, G3BP1 (green arrows) and IMP1 (red arrows), exhibit markedly different SG–cytosol interconversion dynamics. Within granules, both RBPs exhibit alternating binding in the nanocores and liquid droplet-like diffusion in the mobile phase.

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: Binding Assay, Diffusion-based Assay

    Effect of G3BP1 and IMP1 deletions on protein exchange between SGs. (A and B) FDAP curves for PAGFP-G3BP1 C (A) and PAGFP-IMP1 C (B; mean ± SEM, n = 22 and n = 13, respectively). Schematic representations of the deletion constructs are shown on top. Protein interaction (gray) and RNA-binding domains (black) are indicated. Residence time of PAGFP-tagged G3BP1 C (A) and PAGFP-IMP1 C (B) in granules as determined by the model FDAP function (mean ± SEM, n = 22 from four and n = 13 from six independent experiments, respectively) are shown on the right. Values for full-length PAGFP-G3BP1 and PAGFP-IMP1 from Fig. 1 are indicated for comparison by dotted lines (green and red, respectively). The deletion constructs were not showing statistically significant differences to their respective full-length counterparts, but were statistically significantly different from the control construct (3×PAGFP vs. PAGFP-G3BP1 C , P

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: Effect of G3BP1 and IMP1 deletions on protein exchange between SGs. (A and B) FDAP curves for PAGFP-G3BP1 C (A) and PAGFP-IMP1 C (B; mean ± SEM, n = 22 and n = 13, respectively). Schematic representations of the deletion constructs are shown on top. Protein interaction (gray) and RNA-binding domains (black) are indicated. Residence time of PAGFP-tagged G3BP1 C (A) and PAGFP-IMP1 C (B) in granules as determined by the model FDAP function (mean ± SEM, n = 22 from four and n = 13 from six independent experiments, respectively) are shown on the right. Values for full-length PAGFP-G3BP1 and PAGFP-IMP1 from Fig. 1 are indicated for comparison by dotted lines (green and red, respectively). The deletion constructs were not showing statistically significant differences to their respective full-length counterparts, but were statistically significantly different from the control construct (3×PAGFP vs. PAGFP-G3BP1 C , P

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: Construct, RNA Binding Assay

    Reduction in the number of LC regions of G3BP1 results in less detectable mobile molecules within the liquid phase without changing binding properties to nanocores. (A) Example of a representative granule after expression of a deletion fragment of G3BP1 (G3BP1 C ) lacking two LC domains fused to HaloTag on a SNAP-IMP1 background. The granule was identified based on the SiR staining (left) and localizations and trajectories based on TMR staining of HaloTag-G3BP1 C (right) are shown. The border of the SG is indicated by a dashed line. Note that most of the trajectories remained locally very restricted. Bar, 1 µm. (B) Probability density of single-molecule diffusion coefficients from trajectories of a granule expressing HaloTag-G3BP1 C showing the presence of a substantial fraction of essentially immobile molecules. (C) Lifetime determination of HaloTag-G3BP1 C on SNAP-IMP1 background in the bound fraction of SGs. Quantification of granules from six cells from two independent experiments was performed. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: Reduction in the number of LC regions of G3BP1 results in less detectable mobile molecules within the liquid phase without changing binding properties to nanocores. (A) Example of a representative granule after expression of a deletion fragment of G3BP1 (G3BP1 C ) lacking two LC domains fused to HaloTag on a SNAP-IMP1 background. The granule was identified based on the SiR staining (left) and localizations and trajectories based on TMR staining of HaloTag-G3BP1 C (right) are shown. The border of the SG is indicated by a dashed line. Note that most of the trajectories remained locally very restricted. Bar, 1 µm. (B) Probability density of single-molecule diffusion coefficients from trajectories of a granule expressing HaloTag-G3BP1 C showing the presence of a substantial fraction of essentially immobile molecules. (C) Lifetime determination of HaloTag-G3BP1 C on SNAP-IMP1 background in the bound fraction of SGs. Quantification of granules from six cells from two independent experiments was performed. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: Binding Assay, Expressing, Staining, Diffusion-based Assay, Imaging

    G3BP1 and IMP1 exhibit a biphasic partition in a bound and mobile fraction within SGs. (A) Example of a representative granule in a double-transfected cell. The granule was identified based on SiR staining (left) and localizations and trajectories based on TMR staining of HaloTag-G3BP1 (middle and right) are shown. The border of the SG is indicated by a dashed line. Bars, 1 µm. (B) Trajectory of a single G3BP1 molecule (as indicated by an arrowhead at time point 0) showing alternating phases of fast movement and immobile time periods. The trajectory is indicated in green. Bar, 250 nm. (C) High-mobility trajectories (mobile fraction, left) and low mobility trajectories (bound fraction, right) of HaloTag-G3BP1 within the granule shown in A. Bar, 1 µm. (D) Probability density of single-molecule diffusion coefficients from trajectories of a single granule expressing HaloTag-G3BP1 showing a biphasic partition in a bound (red) and mobile fraction (green). (E) Quantification of bound fractions of the respective Halo-tagged constructs expressed in different combinations. Calculations were performed on the total trajectories of 18–28 SGs per experimental condition as indicated by “n.” SGs were obtained from 12–15 cells from 3–10 independent experiments. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: G3BP1 and IMP1 exhibit a biphasic partition in a bound and mobile fraction within SGs. (A) Example of a representative granule in a double-transfected cell. The granule was identified based on SiR staining (left) and localizations and trajectories based on TMR staining of HaloTag-G3BP1 (middle and right) are shown. The border of the SG is indicated by a dashed line. Bars, 1 µm. (B) Trajectory of a single G3BP1 molecule (as indicated by an arrowhead at time point 0) showing alternating phases of fast movement and immobile time periods. The trajectory is indicated in green. Bar, 250 nm. (C) High-mobility trajectories (mobile fraction, left) and low mobility trajectories (bound fraction, right) of HaloTag-G3BP1 within the granule shown in A. Bar, 1 µm. (D) Probability density of single-molecule diffusion coefficients from trajectories of a single granule expressing HaloTag-G3BP1 showing a biphasic partition in a bound (red) and mobile fraction (green). (E) Quantification of bound fractions of the respective Halo-tagged constructs expressed in different combinations. Calculations were performed on the total trajectories of 18–28 SGs per experimental condition as indicated by “n.” SGs were obtained from 12–15 cells from 3–10 independent experiments. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: Transfection, Staining, Diffusion-based Assay, Expressing, Construct, Imaging

    G3BP1 and IMP1 are enriched in distributed nanocores within SGs. (A) Single-molecule imaging of SiR-labeled SNAP-G3BP1 and TMR-labeled HaloTag-IMP1. Snapshots before image acquisition confirmed the presence of SGs and showed that G3BP1 and IMP1 colocalized in the same granules (left). The outline of the cell and the nucleus are indicated. Localizations of single molecules in SGs of the indicated region (yellow box in the snapshot images) are shown (right). Bars: (left, middle) 10 µm; (right) 1 µm. (B) Spatial clustering of single-molecule localizations of an area within a single SG after combined expression of G3BP1 and IMP1. Raw localizations (left), the respective cluster patterns (middle), and cluster pattern overlap (right) are shown. (C) Quantitation of the fraction of the granular area, which is occupied by clusters, and weighted overlap for different combinations of expressed RBPs. The value for “n” represents the number of individual granules analyzed. Granules were obtained from 5–19 cells from three to eight independent experiments. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: G3BP1 and IMP1 are enriched in distributed nanocores within SGs. (A) Single-molecule imaging of SiR-labeled SNAP-G3BP1 and TMR-labeled HaloTag-IMP1. Snapshots before image acquisition confirmed the presence of SGs and showed that G3BP1 and IMP1 colocalized in the same granules (left). The outline of the cell and the nucleus are indicated. Localizations of single molecules in SGs of the indicated region (yellow box in the snapshot images) are shown (right). Bars: (left, middle) 10 µm; (right) 1 µm. (B) Spatial clustering of single-molecule localizations of an area within a single SG after combined expression of G3BP1 and IMP1. Raw localizations (left), the respective cluster patterns (middle), and cluster pattern overlap (right) are shown. (C) Quantitation of the fraction of the granular area, which is occupied by clusters, and weighted overlap for different combinations of expressed RBPs. The value for “n” represents the number of individual granules analyzed. Granules were obtained from 5–19 cells from three to eight independent experiments. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: Imaging, Labeling, Expressing, Quantitation Assay

    Nanocores are relatively immobile within SGs and contain multiple binding sites. (A and B) Top view (left) and kymographic representation (right) of a typical single binding event of Halo-IMP1 on SNAP-G3BP1 background (A) and Halo-G3BP1 on SNAP-IMP1 background (B) demonstrating spatial restriction of nanocores within SGs. (C) Bar plots showing the jump distance distributions of G3BP1 and IMP1 in different combinations indicating the presence of multiple binding sites within a nanocore. 14–27 transition events for each protein per condition were analyzed and the jump distance distributions determined by calculating the pairwise distances of all linked cluster centers. The box represents 50% of the population, whiskers range from 5% to 95%, and the horizontal line shows the median value.

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: Nanocores are relatively immobile within SGs and contain multiple binding sites. (A and B) Top view (left) and kymographic representation (right) of a typical single binding event of Halo-IMP1 on SNAP-G3BP1 background (A) and Halo-G3BP1 on SNAP-IMP1 background (B) demonstrating spatial restriction of nanocores within SGs. (C) Bar plots showing the jump distance distributions of G3BP1 and IMP1 in different combinations indicating the presence of multiple binding sites within a nanocore. 14–27 transition events for each protein per condition were analyzed and the jump distance distributions determined by calculating the pairwise distances of all linked cluster centers. The box represents 50% of the population, whiskers range from 5% to 95%, and the horizontal line shows the median value.

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: Binding Assay

    G3BP1 and IMP1 exhibit different dynamics of protein exchange between SGs. (A) Protein interaction (gray) and RNA-binding domains (black) of human G3BP1 and IMP1 according to SMART analysis for identification of signaling domains ( Schultz et al., 1998 ). RRM, RNA recognition motif; KH, K homology domain. Right: Western analysis of cellular lysates after transfection with PAGFP-tagged G3BP1 and IMP1 (arrowhead). Lysates were analyzed using size-based capillary electrophoresis, and electropherograms are represented as pseudoblots as described in Materials and methods. Molecular mass standards are indicated. The respective endogenous proteins are indicated by an arrow. Please note that analysis by size-based capillary electrophoresis can yield protein mobilities that differ from separation by standard SDS-PAGE. (B) Colocalization of exogenously expressed mCherry-G3BP1 with the SG marker TIA-1. Bar, 10 µm. (C) Colocalization and dynamic exchange of fluorescence-tagged G3BP1 and IMP1 in SGs of living PC12 cells. Granules were labeled with mCherry-IMP1 and PAGFP-G3BP1 was activated in one granule (dashed square). The outline of the cell and the nucleus are indicated in the red fluorescent micrographs. Fluorescence distribution was followed over time. After some seconds, photoactivated PAGFP-G3BP1 appears in SGs outside of the activated region indicating dynamic exchange of G3BP1 between SGs. Bar, 10 µm. (D) Bar plot showing the size distribution of SGs as determined from the area of mCherry-IMP1 positive granules. The box represents 50% of the population, whiskers range from 5% to 95% and crosses correspond to the minimal and maximal values ( n = 34). (E) Schematic representation showing the FDAP approach to determine dynamics and binding of PAGFP-tagged G3BP1 and IMP1 in granules. Photoactivation and fluorescence recording was performed in a 3 × 5 µm region containing cytosol and granules between nucleus and plasma membrane (red box). The decay curves were fitted with model FDAP functions. (F) FDAP curves for PAGFP-IMP1, PAGFP-G3BP1, and 3×PAGFP (mean ± SEM, n = 13 [PAGFP-IMP1], n = 17 [PAGFP-G3BP1], n = 20 [3×PAGFP]) showing the different dynamics of IMP1 and G3BP1. (G) Residence time of PAGFP-tagged IMP1 and G3BP1 in granules as determined by the model FDAP function (mean ± SEM, n = 19 [PAGFP-IMP1], n = 20 [PAGFP-G3BP1], n = 20 [3×PAGFP] from two [3×PAGFP], four [PAGFP-G3BP1], and five [PAGFP-IMP1] independent experiments). Comparison between the constructs involved one-way ANOVA followed by post-hoc Tukey's test. **, P

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: G3BP1 and IMP1 exhibit different dynamics of protein exchange between SGs. (A) Protein interaction (gray) and RNA-binding domains (black) of human G3BP1 and IMP1 according to SMART analysis for identification of signaling domains ( Schultz et al., 1998 ). RRM, RNA recognition motif; KH, K homology domain. Right: Western analysis of cellular lysates after transfection with PAGFP-tagged G3BP1 and IMP1 (arrowhead). Lysates were analyzed using size-based capillary electrophoresis, and electropherograms are represented as pseudoblots as described in Materials and methods. Molecular mass standards are indicated. The respective endogenous proteins are indicated by an arrow. Please note that analysis by size-based capillary electrophoresis can yield protein mobilities that differ from separation by standard SDS-PAGE. (B) Colocalization of exogenously expressed mCherry-G3BP1 with the SG marker TIA-1. Bar, 10 µm. (C) Colocalization and dynamic exchange of fluorescence-tagged G3BP1 and IMP1 in SGs of living PC12 cells. Granules were labeled with mCherry-IMP1 and PAGFP-G3BP1 was activated in one granule (dashed square). The outline of the cell and the nucleus are indicated in the red fluorescent micrographs. Fluorescence distribution was followed over time. After some seconds, photoactivated PAGFP-G3BP1 appears in SGs outside of the activated region indicating dynamic exchange of G3BP1 between SGs. Bar, 10 µm. (D) Bar plot showing the size distribution of SGs as determined from the area of mCherry-IMP1 positive granules. The box represents 50% of the population, whiskers range from 5% to 95% and crosses correspond to the minimal and maximal values ( n = 34). (E) Schematic representation showing the FDAP approach to determine dynamics and binding of PAGFP-tagged G3BP1 and IMP1 in granules. Photoactivation and fluorescence recording was performed in a 3 × 5 µm region containing cytosol and granules between nucleus and plasma membrane (red box). The decay curves were fitted with model FDAP functions. (F) FDAP curves for PAGFP-IMP1, PAGFP-G3BP1, and 3×PAGFP (mean ± SEM, n = 13 [PAGFP-IMP1], n = 17 [PAGFP-G3BP1], n = 20 [3×PAGFP]) showing the different dynamics of IMP1 and G3BP1. (G) Residence time of PAGFP-tagged IMP1 and G3BP1 in granules as determined by the model FDAP function (mean ± SEM, n = 19 [PAGFP-IMP1], n = 20 [PAGFP-G3BP1], n = 20 [3×PAGFP] from two [3×PAGFP], four [PAGFP-G3BP1], and five [PAGFP-IMP1] independent experiments). Comparison between the constructs involved one-way ANOVA followed by post-hoc Tukey's test. **, P

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: RNA Binding Assay, Western Blot, Transfection, Electrophoresis, SDS Page, Marker, Fluorescence, Labeling, Binding Assay, Construct

    G3BP1 and IMP1 have a short lifetime in the bound fraction and display anomalous subdiffusion in the mobile fraction. (A) Lifetime determination of different combinations of HaloTag- and SNAP-tagged G3BP1 and IMP1 in the bound fraction of SGs. Trajectories of the respective Halo-tagged constructs (shown in bold) are evaluated. (B) Example of the trajectories of the mobile fraction of HaloTag-G3BP1 within a single granule (left) and plot of the MSD against the elapsed time showing anomalous diffusion. Mean diffusion constants (Γ) and α values of the respective HaloTag construct in the mobile fraction as determined for the different combinations. Bar, 500 nm. Calculations were performed on the mobile trajectories of 8–11 SGs per experimental condition as indicated by n. SGs were obtained from 8–11 cells from two to eight independent experiments. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Journal: The Journal of Cell Biology

    Article Title: Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

    doi: 10.1083/jcb.201709007

    Figure Lengend Snippet: G3BP1 and IMP1 have a short lifetime in the bound fraction and display anomalous subdiffusion in the mobile fraction. (A) Lifetime determination of different combinations of HaloTag- and SNAP-tagged G3BP1 and IMP1 in the bound fraction of SGs. Trajectories of the respective Halo-tagged constructs (shown in bold) are evaluated. (B) Example of the trajectories of the mobile fraction of HaloTag-G3BP1 within a single granule (left) and plot of the MSD against the elapsed time showing anomalous diffusion. Mean diffusion constants (Γ) and α values of the respective HaloTag construct in the mobile fraction as determined for the different combinations. Bar, 500 nm. Calculations were performed on the mobile trajectories of 8–11 SGs per experimental condition as indicated by n. SGs were obtained from 8–11 cells from two to eight independent experiments. For all experiments, stress had been induced by a 20-min treatment with 0.5 mM sodium arsenite before imaging.

    Article Snippet: The following antibodies were used: anti–TIA-1 (G-3; mouse monoclonal; Santa Cruz Biotechnology), anti-G3BP1 (NBP2; rabbit polyclonal; Novus Biologicals), anti-IMP1 (D-9; mouse monoclonal; Santa Cruz Biotechnology).

    Techniques: Construct, Diffusion-based Assay, Imaging

    Association of CD38 protein expression with prostate cancer recurrence. a Representative images of CD38 staining in normal and cancer cores from the PSA progression tissue microarray. Scale bars represent 100 μm. b Waterfall plot representing composite CD38 staining scores in normal and cancer cores scaled to the mean of all values. c Box plot of composite CD38 staining scores. Statistics represent Welch Two Sample t test. d Kaplan-Meier plot of biochemical recurrence-free survival for patients with CD38 staining scores greater than the median in both normal and cancer cores compared to all other patients (remainder). Breslow test p value is shown, with a hazard ratio of 0.71

    Journal: Cancer & Metabolism

    Article Title: CD38 is methylated in prostate cancer and regulates extracellular NAD+

    doi: 10.1186/s40170-018-0186-3

    Figure Lengend Snippet: Association of CD38 protein expression with prostate cancer recurrence. a Representative images of CD38 staining in normal and cancer cores from the PSA progression tissue microarray. Scale bars represent 100 μm. b Waterfall plot representing composite CD38 staining scores in normal and cancer cores scaled to the mean of all values. c Box plot of composite CD38 staining scores. Statistics represent Welch Two Sample t test. d Kaplan-Meier plot of biochemical recurrence-free survival for patients with CD38 staining scores greater than the median in both normal and cancer cores compared to all other patients (remainder). Breslow test p value is shown, with a hazard ratio of 0.71

    Article Snippet: In contrast to the prostate, seminal vesicle epithelial cells express high levels of CD38 and this expression is abolished in knockout mice (Fig. ).

    Techniques: Expressing, Staining, Microarray

    CD38 regulates extracellular but not intracellular NAD + levels in RPWE1 cells. a Western blots demonstrate doxycyline (Dox) induced expression of wild-type (WT) or mutant (E226Q) CD38 in RWPE1 cells. Tubulin serves as a loading control. b Cell proliferation assay over 4 days in culture in the presence or absence of 20 ng/mL Dox. Relative cell number was assessed by measuring DNA fluorescence at 465 nm. 3–6 replicate wells per group per time point were measured. Plot shows mean ± standard error of the mean (SEM). c , d NAD + and NADH levels were measured relative to total protein in each sample and presented relative to no Dox (non-induced) sample. Mean ± SEM of four replicates is shown. e NAD + :NADH ratio is calculated based on results shown in c and d . Mean ± SEM of four replicates is shown. f Cells were treated with Triton X-100 (TX-100) to permeabilize cells followed by NAD + measurements. NAD + /protein is shown relative to no Dox. Mean ± SEM of four replicates is shown. g – i RWPE1 cells were treated with increasing concentrations of FK866 followed by NAD + ( g ) and NADH ( h ) measurements. Mean ± SEM of four replicates is shown. Newman-Keuls Multiple Comparison Test. i Cell proliferation assay over 4 days in culture in the presence of the indicated concentrations of FK866. DNA fluorescence represents relative cell number. 3–6 replicate wells per group per time point were measured. Plot shows mean ± standard error of the mean (SEM). j Relative NAD + /protein levels in the media 30 min after the addition of 800 nM exogenous NAD + . Mean ± SEM of four replicates is shown

    Journal: Cancer & Metabolism

    Article Title: CD38 is methylated in prostate cancer and regulates extracellular NAD+

    doi: 10.1186/s40170-018-0186-3

    Figure Lengend Snippet: CD38 regulates extracellular but not intracellular NAD + levels in RPWE1 cells. a Western blots demonstrate doxycyline (Dox) induced expression of wild-type (WT) or mutant (E226Q) CD38 in RWPE1 cells. Tubulin serves as a loading control. b Cell proliferation assay over 4 days in culture in the presence or absence of 20 ng/mL Dox. Relative cell number was assessed by measuring DNA fluorescence at 465 nm. 3–6 replicate wells per group per time point were measured. Plot shows mean ± standard error of the mean (SEM). c , d NAD + and NADH levels were measured relative to total protein in each sample and presented relative to no Dox (non-induced) sample. Mean ± SEM of four replicates is shown. e NAD + :NADH ratio is calculated based on results shown in c and d . Mean ± SEM of four replicates is shown. f Cells were treated with Triton X-100 (TX-100) to permeabilize cells followed by NAD + measurements. NAD + /protein is shown relative to no Dox. Mean ± SEM of four replicates is shown. g – i RWPE1 cells were treated with increasing concentrations of FK866 followed by NAD + ( g ) and NADH ( h ) measurements. Mean ± SEM of four replicates is shown. Newman-Keuls Multiple Comparison Test. i Cell proliferation assay over 4 days in culture in the presence of the indicated concentrations of FK866. DNA fluorescence represents relative cell number. 3–6 replicate wells per group per time point were measured. Plot shows mean ± standard error of the mean (SEM). j Relative NAD + /protein levels in the media 30 min after the addition of 800 nM exogenous NAD + . Mean ± SEM of four replicates is shown

    Article Snippet: In contrast to the prostate, seminal vesicle epithelial cells express high levels of CD38 and this expression is abolished in knockout mice (Fig. ).

    Techniques: Western Blot, Expressing, Mutagenesis, Proliferation Assay, Fluorescence

    The CD38 locus undergoes CpG hypermethylation in prostate cancer. a Schematic representation of the CD38 locus. Note that the CpG island (green box) extends downstream of the transcriptional start site into the first intron. In silico analysis of publicly available reduced representation bisulfite sequencing (RRBS) is shown below (heat map: red—dense methylation, green—no methylation). The location of the PCR amplicon used in subsequent COMPARE-MS experiments is indicated by the red box. b – e Methylation heat maps derived from COMPARE-MS analysis of benign prostate tissue ( b ), primary prostate cancers ( c ), and prostate cancer metastases ( d , e ) show hypermethylation of the CD38 in primary prostate cancer and prostate cancer metastases (heat map: red—dense methylation; white—no methylation). f In silico analysis of TCGA data reveals frequent hypermethylation of the CD38 locus. Correlation plots of log2 mRNA expression (based on RNA-seq, RSEM z-scores) and methylation levels (based on Infinium Human Methylation 450k BeadChip analysis) in 333 primary prostate cancer samples

    Journal: Cancer & Metabolism

    Article Title: CD38 is methylated in prostate cancer and regulates extracellular NAD+

    doi: 10.1186/s40170-018-0186-3

    Figure Lengend Snippet: The CD38 locus undergoes CpG hypermethylation in prostate cancer. a Schematic representation of the CD38 locus. Note that the CpG island (green box) extends downstream of the transcriptional start site into the first intron. In silico analysis of publicly available reduced representation bisulfite sequencing (RRBS) is shown below (heat map: red—dense methylation, green—no methylation). The location of the PCR amplicon used in subsequent COMPARE-MS experiments is indicated by the red box. b – e Methylation heat maps derived from COMPARE-MS analysis of benign prostate tissue ( b ), primary prostate cancers ( c ), and prostate cancer metastases ( d , e ) show hypermethylation of the CD38 in primary prostate cancer and prostate cancer metastases (heat map: red—dense methylation; white—no methylation). f In silico analysis of TCGA data reveals frequent hypermethylation of the CD38 locus. Correlation plots of log2 mRNA expression (based on RNA-seq, RSEM z-scores) and methylation levels (based on Infinium Human Methylation 450k BeadChip analysis) in 333 primary prostate cancer samples

    Article Snippet: In contrast to the prostate, seminal vesicle epithelial cells express high levels of CD38 and this expression is abolished in knockout mice (Fig. ).

    Techniques: In Silico, Methylation Sequencing, Methylation, Polymerase Chain Reaction, Amplification, Mass Spectrometry, Derivative Assay, Expressing, RNA Sequencing Assay

    Reduced expression of CD38 in metastatic prostate cancer. a Waterfall plot indicating relative CD38 mRNA expression from RNA sequencing of benign prostate, localized prostate cancer, metastatic castration-resistant prostate cancer (CRPC) with an adenocarcinoma (adeno) phenotype and metastatic CRPC with a neuroendocrine prostate cancer (NEPC) phenotype. Expression levels are presented as Log of FPKM-1 (Fragments Per Kilobase Million) values and scaled to the mean of all values shown. b Box plot corresponding to samples from a presented as Log of FPKM values

    Journal: Cancer & Metabolism

    Article Title: CD38 is methylated in prostate cancer and regulates extracellular NAD+

    doi: 10.1186/s40170-018-0186-3

    Figure Lengend Snippet: Reduced expression of CD38 in metastatic prostate cancer. a Waterfall plot indicating relative CD38 mRNA expression from RNA sequencing of benign prostate, localized prostate cancer, metastatic castration-resistant prostate cancer (CRPC) with an adenocarcinoma (adeno) phenotype and metastatic CRPC with a neuroendocrine prostate cancer (NEPC) phenotype. Expression levels are presented as Log of FPKM-1 (Fragments Per Kilobase Million) values and scaled to the mean of all values shown. b Box plot corresponding to samples from a presented as Log of FPKM values

    Article Snippet: In contrast to the prostate, seminal vesicle epithelial cells express high levels of CD38 and this expression is abolished in knockout mice (Fig. ).

    Techniques: Expressing, RNA Sequencing Assay

    CD38 regulates extracellular NAD + levels in mouse tissues. a Flow cytometry histogram plots gated on EpCAM+ epithelial cells from wild-type (CD38+/+) or knockout (CD38−/−) prostate and seminal vesicle cells and stained for surface expression of CD38. b , c NAD + and NADH levels were measured using the NAD + /NADH-Glo assay normalized to total DNA in each tissue and presented relative to wild-type. Mean ± SEM of four replicates is shown. d , e NAD + levels in intact seminal vesicle tissue ( d ) or in tissue after having removed the fluid ( e ) normalized to protein and presented relative to wild-type. Mean ± SEM of 2–5 replicates is shown. f NAD + levels remaining in the media were measured 60 min after the addition of 800 nM exogenous NAD + to purified seminal vesicle cells. Fold reduction in initial NAD + levels, relative to wild-type, is shown. Mean ± SEM of duplicates is shown. g Relative intracellular NAD + levels from wild-type seminal vesicle cells measured 60 min after the addition of 0 or 800 nM NAD + . Mean ± SEM of three replicates is shown

    Journal: Cancer & Metabolism

    Article Title: CD38 is methylated in prostate cancer and regulates extracellular NAD+

    doi: 10.1186/s40170-018-0186-3

    Figure Lengend Snippet: CD38 regulates extracellular NAD + levels in mouse tissues. a Flow cytometry histogram plots gated on EpCAM+ epithelial cells from wild-type (CD38+/+) or knockout (CD38−/−) prostate and seminal vesicle cells and stained for surface expression of CD38. b , c NAD + and NADH levels were measured using the NAD + /NADH-Glo assay normalized to total DNA in each tissue and presented relative to wild-type. Mean ± SEM of four replicates is shown. d , e NAD + levels in intact seminal vesicle tissue ( d ) or in tissue after having removed the fluid ( e ) normalized to protein and presented relative to wild-type. Mean ± SEM of 2–5 replicates is shown. f NAD + levels remaining in the media were measured 60 min after the addition of 800 nM exogenous NAD + to purified seminal vesicle cells. Fold reduction in initial NAD + levels, relative to wild-type, is shown. Mean ± SEM of duplicates is shown. g Relative intracellular NAD + levels from wild-type seminal vesicle cells measured 60 min after the addition of 0 or 800 nM NAD + . Mean ± SEM of three replicates is shown

    Article Snippet: In contrast to the prostate, seminal vesicle epithelial cells express high levels of CD38 and this expression is abolished in knockout mice (Fig. ).

    Techniques: Flow Cytometry, Cytometry, Knock-Out, Staining, Expressing, Glo Assay, Purification