nelf  (Bethyl)

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Article Snippet: NELFE , 1 μg/mL , Rabbit , Monoclonal , A301-913A , Bethyl laboratories.

Techniques: Combined Bisulfite Restriction Analysis Assay

Journal: Scientific Reports

Article Title: Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

doi: 10.1038/s41598-021-92806-8

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Article Snippet: NELFE , 1 μg/mL , Rabbit , Monoclonal , A301-913A , Bethyl laboratories.

Techniques: Combined Bisulfite Restriction Analysis Assay

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: ( A ) The four-subunit human NELF complex was recombinantly expressed in insect cells and purified to homogeneity. The purified complex (0.9 µg) was run on a 4–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. A molecular weight marker (MW) in kilodaltons (kDa) is provided on the left side of the gel. ( B ) Cartoon depiction of individual NELF proteins. Different shades designate unique regions or domains of each protein (NELF-A red, NELF-B orange, NELF-C cyan, NELF-E purple). N- and C-term refers to the N- or C-terminus of a protein, respectively. RD repeats refer to a flexible region of NELF-E that is primarily composed of Arg and Asp residues. ( C – H ) Crosslinks detected within the NELF tetramer displayed as binary interactions. Intraprotein crosslinks are shown as curves and colored as in ( B ). Interprotein crosslinks are shown as black lines. The endpoint of each line specifies a specific residue in the corresponding protein. Crosslinking data is filtered to display crosslinks with an ID-score greater than 30. A full map of all interprotein crosslinks is provided in . All crosslinks can be found in . Crosslinking data modeled with xiNET . ( C ) NELF-A, NELF-C ( D ) NELF-B, NELF-A ( E ) NELF-B, NELF-C ( F ) NELF-B, NELF-E ( G ) NELF-E, NELF-C ( H ) NELF-E, NELF-A. DOI: http://dx.doi.org/10.7554/eLife.14981.009 10.7554/eLife.14981.010 Figure 3—source data 1. Crosslinking MS of four-subunit NELF complex primary data for intra and interprotein crosslinks. DOI: http://dx.doi.org/10.7554/eLife.14981.010

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Molecular Weight, Marker

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: ( A ) Map of all crosslinks detected between NELF subunits. Crosslinks emanating from NELF-A are red, NELF-C are cyan, and NELF-E are purple. ( B ) Representative spectra of NELF inter and intraprotein crosslinks as determined by mass spectrometry. Sequences for crosslinked peptides are shown above the spectra. ( C ) Distances between inter- and intraprotein crosslinks within the NELF-AC crystal structure. Distances were measured in PyMol ( PyMOL, 2002 ). Crosslinks greater than 30Å can be attributed to a conformation not present in the crystal structure between NELF-C helices 12’ and 13’ α helices 18’ and 19’. ( D ) Model of NELF-B as generated by I-TASSER. The model suggests NELF-B is highly alpha helical in nature and composed of heat repeats. NELF-B lysines that form inter- and intrasubunit crosslinks are indicated on the model. ( E ) Intrasubunit crosslinks between lysines 260 and 332 of the NELF-E RRM are marked on the NMR structure indicating the RRM fold is maintained in context of the NELF tetramer (PDB ID: 2JX2) . DOI: http://dx.doi.org/10.7554/eLife.14981.011

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Mass Spectrometry, Generated

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: Two views of the solvent-accessible surface related by 180° rotations around a vertical axis are shown. ( A ) Surface representation of NELF-AC with shaded ellipses representing regions where NELF-B (orange) or NELF-E (purple) crosslinks with NELF-AC were detected. Lysines are shown in stick representation and numbered. NELF-B and NELF-E primarily crosslink to one face of the NELF-AC dimer. Four empty ellipses represent patches of positively charged residues. ( B ) Surface conservation. Residues that are invariant from human to Drosophila are in green, conserved residues in yellow . Surface areas involved in nucleic acid binding (patches 1–4) (Results) are highlighted. Colors of labels according to color code of protein features belong to . ( C ) Electrostatic surface potential generated with ABPS . Blue, red, and white areas indicate positive, negative and neutral charge, respectively. Surface areas involved in nucleic acid binding (patches 1–4). Colors of labels according to color code of protein features belong to . DOI: http://dx.doi.org/10.7554/eLife.14981.012

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Binding Assay, Generated

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: Curve fitting data for NELF RNA and DNA binding as determined by fluorescence anisotropy. Fluorescence anisotropy data found in Figures 5 – 7 were fit with a single site binding model where possible (Materials and methods). Apparent disassociation constants (K d,app ), R 2 , and Bmax (maximum anisotropy) values with error where applicable for the 60% ssRNA and DNA substrate as well as the TAR RNA are shown. NA means fitting was not applicable. DOI: http://dx.doi.org/10.7554/eLife.14981.016

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Binding Assay, Fluorescence, Construct, Mutagenesis

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: ( A ) NELF constructs expressed and purified from insect cells represented as cartoons. NELF was produced as a FL construct, a construct lacking the NELF-E RRM (NELF ∆RRM), or without NELF-E (NELF-ABC). ( B ) Binding of the WT NELF tetramer, NELF ∆RRM, or NELF-ABC to 10 nM of the 60% GC content RNA as determined by relative change in fluorescence anisotropy. Data was fit with a single site binding equation when possible. Apparent Kd values are found in . Error bars reflect the standard deviation from three experimental replicates. ( C–E ) Binding of the WT or patch mutated NELF-C variants to 10 nM of the 60% GC content RNA as determined by fluorescence anisotropy. The following residues were mutated in the NELF-C patch mutant: R291Q, K315M, K371M, K372M, K374M, K384M, K388M, R419Q, R506Q. Darker shades of red indicate the WT protein constructs whereas lighter shades of red indicate the NELF-C patch mutated variant. Error bars reflect the standard deviation from three experimental replicates. When possible, curves were fit with a single site binding model and apparent Kd values are found in . ( C ) NELF tetramer ( D ) NELF ∆RRM ( E ) NELF-ABC. DOI: http://dx.doi.org/10.7554/eLife.14981.017

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Construct, Purification, Produced, Binding Assay, Fluorescence, Standard Deviation, Mutagenesis, Variant Assay

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: ( A ) Immunoprecipitation of the entire NELF complex by a NELF-E specific antibody from Jurkat cells treated with 4-thiouridine (4sU) for 16 hr and UV-crosslinked as detected by Western blot anaylsis with subunit specific antibodies. Cells treated with 4sU, but not UV-crosslinked are shown as a control. Star indicates NELF-C detected on the same blot. Western blot detected with HRP-conjugated secondary antibodies. ( B ) Phosphorimage of SDS-PAGE used to resolve RNAs crosslinked to NELF subunits from Panel A. Protein crosslinked to RNA was treated with PNK in the presence of ATP [γ-32P]. The resulting protein was run on a 4–12% gradient SDS-PAGE. The gel was incubated with a phosphorimage screen and imaged with a Typhoon imager. In crosslinked cells, a band is seen for the NELF-E and NELF-B/C subunits. Cells treated with 4sU, but not UV-crosslinked are shown as a control. A Coomassie stained gel with purified NELF is shown as size reference. ( C ) Phosphorimage of SDS-PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to overexpressed -C (left), or -B (right) 293FT cells. Overexpressed NELF-C/-B were immunoprecipitated by N-terminal 3X FLAG tags. Coomassie stained gels of overexpressed NELF-C and -B are shown as a size reference. See for further controls. DOI: http://dx.doi.org/10.7554/eLife.14981.020

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Immunoprecipitation, Western Blot, SDS Page, Incubation, Staining, Purification, Labeling

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: ( A ) Expression of NELF subunits in Jurkat and 293FT cells as determined by Western blot analysis. GAPDH was used as loading control. ( B ) The entire NELF complex is immunoprecipitated from 293FT cells by a NELF-E specific antibody. Cells were treated for 16 hr with 4sU prior to UV crosslinking and immunoprecipitaiton. Western blot analysis was performed with samples taken before and after NELF-E immunoprecipitation. Star indicates the NELF-C antibody. The same membrane was reblotted and not stripped between antibody applications. ( C ) Phosphorimage of SDS-PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to NELF subunits from Panel B. Immunoprecipitated samples from cells treated with 4sU but not UV-crosslinked are shown as a control. Purified NELF subjected to SDS-PAGE and Coomassie staining is shown as a size reference. ( D ) Immunoprecipitation of NELF from Jurkat cells by a NELF-A specific antibody. Cells were treated for 16 hr with 4sU prior to UV crosslinking and immunoprecipitaiton. Western blot analysis was performed with samples taken before and after NELF-A immunoprecipitation. ( E ) Phosphorimage of SDS-PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to NELF subunits derived from panel D. Immunoprecipitated samples from cells treated with 4sU but not UV crosslinked are shown as a control. Purified NELF subjected to SDS-PAGE and Coomassie staining is shown as a size reference. DOI: http://dx.doi.org/10.7554/eLife.14981.021

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Expressing, Western Blot, Immunoprecipitation, SDS Page, Labeling, Purification, Staining, Derivative Assay

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: After immunoprecipitation of NELF from Jurkat cells with a NELF-E antibody, protein was run on a gradient 4–12% SDS-PAGE. Bands corresponding to the molecular weight of NELF subunits were subjected to mass spectrometry to determine the protein identity. Peptides detected are marked on the protein sequence for each NELF subunit in green. Peptide counts and coverage are similar for each subunit, indicating the entire NELF complex is pulled down by the NELF-E antibody. DOI: http://dx.doi.org/10.7554/eLife.14981.022

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: Immunoprecipitation, SDS Page, Molecular Weight, Mass Spectrometry, Sequencing

Journal: eLife

Article Title: Architecture and RNA binding of the human negative elongation factor

doi: 10.7554/eLife.14981

Figure Lengend Snippet: The NELF-AC crystal structure, crosslinking data, and in vivo experiments reveal the overall architecture of NELF. NELF-AC (NELF-A red, NELF-C cyan) forms a stable complex that interacts with the N-terminus of NELF-B (orange). NELF-B N- and C- termini sandwich the NELF-E N-terminus. The NELF-E RRM is loosely connected to the entire complex. RNA binds to NELF-E, NELF-B, and NELF-C. The two faces of NELF-AC determined by crystallography and crosslinking are shown below the complex in cartoon format. Three RNA binding faces on the surface of NELF-C are marked with numbers (1, 2, 4). The region of NELF-A that is predicted to bind Pol II, which is absent in our crystal structure is boxed. N- and C- termini for each protein are marked. DOI: http://dx.doi.org/10.7554/eLife.14981.024

Article Snippet: For endogenous NELF-A and NELF-E immunoprecipitation experiments, 30 mg of total protein was incubated with 70 μg of anti-NELF-A antibody (Santa Cruz, sc-23599) or 50 μg of anti-NELF-E antibody (Bethyl Laboratories, A301-914A) conjugated to Protein G Dynabeads (Invitrogen, B00262).

Techniques: In Vivo, RNA Binding Assay