guanabenz acetate  (Tocris)

Journal: Nucleic acids research

Article Title: HELZ2: a new, interferon-regulated, human 3'-5' exoribonuclease of the RNB family is expressed from a non-canonical initiation codon.

doi: 10.1093/nar/gkad673

Figure Lengend Snippet: Figure 3. HELZ2 and HELZ2 are acti v e ribonucleases. ( A ) Sequence alignment of the RNB domain catalytic region from human HELZ2 (Q9BYK8), DIS3 (Q9Y2L1), DIS3L1 (Q8TF46), DIS3L2 (Q8IYB7) and from bacterial RNaseII (P30850). Previously proposed catalytic residues are indicated by asterisks. The sequence alignment is coloured for amino acid conservation using Clustal W. The position naturally substituted by an asparagine in human HELZ2 is indicated by a red arrowhead while the aspartate mutated to an asparagine to inactivate the HELZ2 RNB domain (rnb - ) is indicated by a black arrowhead. ( B ) Western-blot using anti-GFP showing e xpression le v els and immunoprecipitation efficiencies for the proteins used in the ribonuclease ac- ti vity test. ( C ) Representati v e RNA degradation assay gel. Samples from the ribonuclease activity test were fractionated on a 20% polyacrylamide-urea gel. Numbers above the lanes r epr esent incubation times in minutes. T1: RNase T1 digestion of the substrate RNA. OH: alkaline hydrolysis of the radiola- belled RNA substrate. Asterisk: non-specific product of the reaction. The slightly stronger background degradation detected with GFP-HELZ2 constructs compared to GFP-MBP likely results from co-precipitation of RNA associated with some contaminating activity with the former. ( D ) Quantification of the bands corresponding to the full-length RNA substrate plotted against the time of the reaction in minutes with standard error ( n = 3).

Article Snippet: Assays with guanabenz acetate RNase and ATPase assays were performed as described in the presence or absence of guanabenz acetate (sc-203590, Santa Cruz).

Techniques: Sequencing, Western Blot, Immunoprecipitation, Degradation Assay, Activity Assay, Incubation, Construct

Journal: Nucleic acids research

Article Title: HELZ2: a new, interferon-regulated, human 3'-5' exoribonuclease of the RNB family is expressed from a non-canonical initiation codon.

doi: 10.1093/nar/gkad673

Figure Lengend Snippet: Figure 4. HELZ2 displays substrate pr efer ence for p yrimidines. ( A ) Repr esentati v e RNA degradation assay gel with oligo(A), oligo(U), oligo(C) or oligo(G) containing substrates. Samples from the ribonuclease activity test were fractionated on a 20% polyacrylamide-urea gel. Numbers above the lanes r epr esent incubation times in minutes. A radiolabelled 10 nucleotide long DN A (DN A 10) was also loaded as r efer ence to determine the sizes of the final degradation products. Asterisk: non-specific product of the reaction. ( B ) Western-blot using anti-GFP showing expression levels and immunoprecipitation efficiency for the proteins used in the ribonuclease activity test. ( C ) Quantification of the bands corresponding to the full-length DN A–RN A substrates plotted against the time of the reaction in minutes with standard error ( n = 3).

Article Snippet: Assays with guanabenz acetate RNase and ATPase assays were performed as described in the presence or absence of guanabenz acetate (sc-203590, Santa Cruz).

Techniques: Degradation Assay, Activity Assay, Incubation, Western Blot, Expressing, Immunoprecipitation

Journal: Nucleic acids research

Article Title: HELZ2: a new, interferon-regulated, human 3'-5' exoribonuclease of the RNB family is expressed from a non-canonical initiation codon.

doi: 10.1093/nar/gkad673

Figure Lengend Snippet: Figure 6. Helicase and ribonuclease activities of HELZ2 synergize for the degradation of structured RNA Representati v e RNA degradation assay of duplex RNA substrates in presence or absence of ATP. Samples from the ribonuclease activity test were fractionated on a 20% polyacrylamide- urea gel. Numbers above the lanes r epr esent incubation times in hours. Above the figure is a schematic representation of the substrates and proteins used for the assay are indicated. T1: RNase T1 digestion of the substrate. OH: alkaline hydrolysis of the radiolabelled RNA substrate. Asterisk: non-specific product of the reaction.

Article Snippet: Assays with guanabenz acetate RNase and ATPase assays were performed as described in the presence or absence of guanabenz acetate (sc-203590, Santa Cruz).

Techniques: Degradation Assay, Activity Assay, Incubation

Journal: Nucleic acids research

Article Title: HELZ2: a new, interferon-regulated, human 3'-5' exoribonuclease of the RNB family is expressed from a non-canonical initiation codon.

doi: 10.1093/nar/gkad673

Figure Lengend Snippet: Figure 7. Somatic alterations of HELZ2 inactivate its RNase activity in some cancer patients. ( A ) Representati v e RNA degradation assay gel from duplicate experiments for the different HELZ2 variants indicated on top. MBP and the rnb - (D1601N) mutant serve as negative controls. Samples from the ribonuclease activity test were fractionated on a 20% polyacrylamide–urea gel. Numbers above the lanes represent incubation times in minutes. T1: RNase T1 digestion of the substrate RNA. OH: alkaline hydrolysis of the radiolabelled RNA substrate. Asterisk: non-specific product of the reaction. ( B ) Representati v e ATPase assay using the different HELZ2 variants indicated on top. Assays were performed in the presence of DN A to stim ulate the ATPase activity. The hel - mutant serves as negative contr ol. Samples fr om the ATPase activity tests were fractionated by thin-layer chromato gra phy (TLC) and detected by autor adiogr aphy. Numbers above the lanes r epr esent incubation times in hours. The fraction of ATP hydrolyzed is indicated below each lane. The last two lanes are references for the migration of ATP and ADP, respecti v ely. ( C ) Western-b lot using anti-GFP showing expression le v els and immunoprecipita tion ef ficiencies for the proteins used in the ribonuclease and ATPase activity tests.

Article Snippet: Assays with guanabenz acetate RNase and ATPase assays were performed as described in the presence or absence of guanabenz acetate (sc-203590, Santa Cruz).

Techniques: Activity Assay, Degradation Assay, Mutagenesis, Incubation, ATPase Assay, Migration, Western Blot, Expressing