Acrylamide Gel Assay:
Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Fisher Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0.45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-Rad), according to the manufacturer’s instructions. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Sigma Aldrich) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-GFP (Thermo Fisher Scientific, A-11122, 1:1000), anti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS (SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP (SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, F3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, 1:1000), anti-DHX36 (Proteintech, 13159–1-AP, 1:500) and anti-βActin (SantaCruz Biotech, sc-69879, 1:1000).
Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition and dynamic of Stress Granules
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0, 45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-rad), according to the manufacturer’s instructions. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Difco skim milk) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-GFP (Thermo Scientific, A-11122, 1:1000), anti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS (SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP (SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, F3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, 1:1000), anti-DHX36 (Proteintech, 13159-1-AP, 1:500).
Article Title: SMaRT lncRNA controls translation of a G-quadruplex-containing mRNA antagonizing the DHX36 helicase.
Article Snippet: Protein concentration was assessed using Bradford Protein Assay (Bio-Rad). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad) according to the manufacturer’s instructions, and proteins were transferred to Immobilon-E PVDF 0.45-lm membrane (Merck-Millipore) at 80 V for 1 h in 1× Towbin Transfer Buffer (25 mM TRIS, 192 mM glycine, 20% methanol). .. Membranes were blocked with 5% non-fat dry milk (Difco skim milk) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-DHX36 (13159-1-AP, Proteintech); anti-MLX (12042-1-AP, Proteintech); anti-MyoD (M-318, Sc760, Santa Cruz Biotechnology); anti-MYOG (F5D, sc-12732, Santa Cruz Biotechnology); anti-caspase 3 (9622, Cell Signaling Technology); anti-ACTININ-4 (G-4, sc-390205, Santa Cruz Biotechnology); anti-DYS (NCL-DYS1, Novocastra Laboratories), anti-MEF2C (sc365862, Santa Cruz Biotechnology), anti-GAPDH (6C5, sc-32233, Santa Cruz Biotechnology), and anti-HPRT (FL-218, sc-20975, Santa Cruz Biotechnology).
Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules.
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Fisher Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0.45 μm nitrocellulose membrane, using the TransBlot Turbo S t 5 b i a ( ( F 1 a o p W t ( P F A w C w A b t w p t 1 u I s C d 1 P t a p P f G F 1 T t t w R d P l 3 a w l s i I t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae942/7874844 by guest on 07 N ovem ber 2024 ystem (Bio-Rad), according to the manufacturer’s instrucions. .. Membranes were stained with Ponceau, blocked with % non-fat dry milk (Sigma Aldrich) for 1 h and incuated overnight at 4 ◦C with the following primary antibodes: anti-GFP (Thermo Fisher Scientific, A-11122, 1:1000), nti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, 3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, :1000), anti-DHX36 (Proteintech, 13159–1-AP, 1:500) and nti- βActin (SantaCruz Biotech, sc-69879, 1:1000).
Article Title: Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation
Article Snippet: Protein concentration was assessed by PierceTM 660 nm protein assay (Thermo Fisher Scientific), according to the manufacturer’s instructions. .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0,45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-Rad), according to the manufacturer’s protocol. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Sigma Aldrich) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-RAB7a (Abcam, AB137029, 1:1000), anti-LAMP1 (DHSB biology, H4A3, 1:1000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc-47724, 1:1000), anti-FLAG (Merck, F7425, 1:1000), anti-GEMIN5 (Sigma Aldrich, HPA037393, 1:500), anti-RAB5 (BD Transduction LaboratoriesTM, 610724, 1:1000), anti-H3 (Active motif, AB_2687567, 1C8B2, 1:1000), anti-EEA1 (eBioscienceTM, 14-9114, 1:1000), anti-TOM20 (Santa Cruz Biotechnology, sc-17764, 1:1000), anti-VINCULIN (ProteinTech, 26520-1-AP, 1:5000).
Protein Electrophoresis:Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Fisher Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0.45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-Rad), according to the manufacturer’s instructions. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Sigma Aldrich) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-GFP (Thermo Fisher Scientific, A-11122, 1:1000), anti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS (SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP (SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, F3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, 1:1000), anti-DHX36 (Proteintech, 13159–1-AP, 1:500) and anti-βActin (SantaCruz Biotech, sc-69879, 1:1000).
Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition and dynamic of Stress Granules
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0, 45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-rad), according to the manufacturer’s instructions. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Difco skim milk) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-GFP (Thermo Scientific, A-11122, 1:1000), anti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS (SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP (SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, F3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, 1:1000), anti-DHX36 (Proteintech, 13159-1-AP, 1:500).
Article Title: SMaRT lncRNA controls translation of a G-quadruplex-containing mRNA antagonizing the DHX36 helicase.
Article Snippet: Protein concentration was assessed using Bradford Protein Assay (Bio-Rad). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad) according to the manufacturer’s instructions, and proteins were transferred to Immobilon-E PVDF 0.45-lm membrane (Merck-Millipore) at 80 V for 1 h in 1× Towbin Transfer Buffer (25 mM TRIS, 192 mM glycine, 20% methanol). .. Membranes were blocked with 5% non-fat dry milk (Difco skim milk) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-DHX36 (13159-1-AP, Proteintech); anti-MLX (12042-1-AP, Proteintech); anti-MyoD (M-318, Sc760, Santa Cruz Biotechnology); anti-MYOG (F5D, sc-12732, Santa Cruz Biotechnology); anti-caspase 3 (9622, Cell Signaling Technology); anti-ACTININ-4 (G-4, sc-390205, Santa Cruz Biotechnology); anti-DYS (NCL-DYS1, Novocastra Laboratories), anti-MEF2C (sc365862, Santa Cruz Biotechnology), anti-GAPDH (6C5, sc-32233, Santa Cruz Biotechnology), and anti-HPRT (FL-218, sc-20975, Santa Cruz Biotechnology).
Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules.
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Fisher Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0.45 μm nitrocellulose membrane, using the TransBlot Turbo S t 5 b i a ( ( F 1 a o p W t ( P F A w C w A b t w p t 1 u I s C d 1 P t a p P f G F 1 T t t w R d P l 3 a w l s i I t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae942/7874844 by guest on 07 N ovem ber 2024 ystem (Bio-Rad), according to the manufacturer’s instrucions. .. Membranes were stained with Ponceau, blocked with % non-fat dry milk (Sigma Aldrich) for 1 h and incuated overnight at 4 ◦C with the following primary antibodes: anti-GFP (Thermo Fisher Scientific, A-11122, 1:1000), nti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, 3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, :1000), anti-DHX36 (Proteintech, 13159–1-AP, 1:500) and nti- βActin (SantaCruz Biotech, sc-69879, 1:1000).
Article Title: Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation
Article Snippet: Protein concentration was assessed by PierceTM 660 nm protein assay (Thermo Fisher Scientific), according to the manufacturer’s instructions. .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0,45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-Rad), according to the manufacturer’s protocol. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Sigma Aldrich) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-RAB7a (Abcam, AB137029, 1:1000), anti-LAMP1 (DHSB biology, H4A3, 1:1000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc-47724, 1:1000), anti-FLAG (Merck, F7425, 1:1000), anti-GEMIN5 (Sigma Aldrich, HPA037393, 1:500), anti-RAB5 (BD Transduction LaboratoriesTM, 610724, 1:1000), anti-H3 (Active motif, AB_2687567, 1C8B2, 1:1000), anti-EEA1 (eBioscienceTM, 14-9114, 1:1000), anti-TOM20 (Santa Cruz Biotechnology, sc-17764, 1:1000), anti-VINCULIN (ProteinTech, 26520-1-AP, 1:5000).
Membrane:Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Fisher Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0.45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-Rad), according to the manufacturer’s instructions. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Sigma Aldrich) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-GFP (Thermo Fisher Scientific, A-11122, 1:1000), anti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS (SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP (SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, F3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, 1:1000), anti-DHX36 (Proteintech, 13159–1-AP, 1:500) and anti-βActin (SantaCruz Biotech, sc-69879, 1:1000).
Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition and dynamic of Stress Granules
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0, 45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-rad), according to the manufacturer’s instructions. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Difco skim milk) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-GFP (Thermo Scientific, A-11122, 1:1000), anti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS (SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP (SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, F3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, 1:1000), anti-DHX36 (Proteintech, 13159-1-AP, 1:500).
Article Title: SMaRT lncRNA controls translation of a G-quadruplex-containing mRNA antagonizing the DHX36 helicase.
Article Snippet: Protein concentration was assessed using Bradford Protein Assay (Bio-Rad). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad) according to the manufacturer’s instructions, and proteins were transferred to Immobilon-E PVDF 0.45-lm membrane (Merck-Millipore) at 80 V for 1 h in 1× Towbin Transfer Buffer (25 mM TRIS, 192 mM glycine, 20% methanol). .. Membranes were blocked with 5% non-fat dry milk (Difco skim milk) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-DHX36 (13159-1-AP, Proteintech); anti-MLX (12042-1-AP, Proteintech); anti-MyoD (M-318, Sc760, Santa Cruz Biotechnology); anti-MYOG (F5D, sc-12732, Santa Cruz Biotechnology); anti-caspase 3 (9622, Cell Signaling Technology); anti-ACTININ-4 (G-4, sc-390205, Santa Cruz Biotechnology); anti-DYS (NCL-DYS1, Novocastra Laboratories), anti-MEF2C (sc365862, Santa Cruz Biotechnology), anti-GAPDH (6C5, sc-32233, Santa Cruz Biotechnology), and anti-HPRT (FL-218, sc-20975, Santa Cruz Biotechnology).
Article Title: ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules.
Article Snippet: Protein concentration was assessed using BCA protein assay (Thermo Fisher Scientific). .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0.45 μm nitrocellulose membrane, using the TransBlot Turbo S t 5 b i a ( ( F 1 a o p W t ( P F A w C w A b t w p t 1 u I s C d 1 P t a p P f G F 1 T t t w R d P l 3 a w l s i I t D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkae942/7874844 by guest on 07 N ovem ber 2024 ystem (Bio-Rad), according to the manufacturer’s instrucions. .. Membranes were stained with Ponceau, blocked with % non-fat dry milk (Sigma Aldrich) for 1 h and incuated overnight at 4 ◦C with the following primary antibodes: anti-GFP (Thermo Fisher Scientific, A-11122, 1:1000), nti-TIAR (BD Transduction, 610352, 1:1000), anti-FUS SantaCruz Biotech, sc-47711, 1:2000), anti-GAPDH-HRP SantaCruz Biotech, sc-47724, 1:1000), anti-FLAG (Merck, 3165, 1:1000), anti-ELAVL1 (SantaCruz Biotech, sc-5261, :1000), anti-DHX36 (Proteintech, 13159–1-AP, 1:500) and nti- βActin (SantaCruz Biotech, sc-69879, 1:1000).
Article Title: Neuronal late endosomes serve as selective RNA hubs disrupted by ALS-linked FUS mutation
Article Snippet: Protein concentration was assessed by PierceTM 660 nm protein assay (Thermo Fisher Scientific), according to the manufacturer’s instructions. .. Protein electrophoresis was performed using 4–15% Mini-PROTEAN TGX Precast acrylamide gel (Bio-Rad), and proteins were transferred to 0,45 μm nitrocellulose membrane, using the TransBlot Turbo System (Bio-Rad), according to the manufacturer’s protocol. .. Membranes were stained with Ponceau, blocked with 5% non-fat dry milk (Sigma Aldrich) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-RAB7a (Abcam, AB137029, 1:1000), anti-LAMP1 (DHSB biology, H4A3, 1:1000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc-47724, 1:1000), anti-FLAG (Merck, F7425, 1:1000), anti-GEMIN5 (Sigma Aldrich, HPA037393, 1:500), anti-RAB5 (BD Transduction LaboratoriesTM, 610724, 1:1000), anti-H3 (Active motif, AB_2687567, 1C8B2, 1:1000), anti-EEA1 (eBioscienceTM, 14-9114, 1:1000), anti-TOM20 (Santa Cruz Biotechnology, sc-17764, 1:1000), anti-VINCULIN (ProteinTech, 26520-1-AP, 1:5000).
Protein Concentration:
Bradford Assay:
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