rabbit anti xrn1  (Bethyl)

Journal: eLife

Article Title: Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress

doi: 10.7554/eLife.96284

Figure Lengend Snippet: ( a ) Immunoblot for XRN1, eIF2α-P, and eIF2α for cells transfected with a non-targeting (siCTRL) or XRN1- targeting (si XRN1 ) siRNA. ACTB is used as control. ( b ) Cumulative distribution plot of transcript length for XRN1 knockdown (si XRN1 ) and control (siCTRL) cells with and without arsenite treatment (unstressed). ( c ) Same as ( b ) but only reads with ligated 5ʹ end adapter are used. ( d ) Box plots of differential transcript length in arsenite-treated versus unstressed cells for significantly and nonsignificantly shortened transcripts upon XRN1 knockdown and control. ( e, f ) Cumulative distribution plot of transcript length for NOT8* D40A E42A and GFP-expressing cells ( e ) or DCP2* E148Q and GFP-expressing cells ( f ) with or without (unstressed) arsenite treatment. Figure 3—source data 1. Original files for western blot analysis displayed in . Figure 3—source data 2. File containing original western blots for , indicating the relevant bands and treatments.

Article Snippet: Antibody , Rabbit-anti XRN1 polyclonal , Thermo Fisher , A300-443A; RRID: AB_2219047 , (1:1000).

Techniques: Western Blot, Transfection, Control, Knockdown, Expressing

Journal: eLife

Article Title: Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress

doi: 10.7554/eLife.96284

Figure Lengend Snippet: ( a ) Immunoblot for XRN1 for cells transfected with non-targeting and XRN1 -targeting siRNA at varying concentrations. ACTB is used as control. ( b ) Visualization by epifluorescence of GFP, GFP-fused DCP2* E148Q and GFP-fused NOT8* D40A E42A expression in HeLa cells. Scale bar 50 µm. ( c ) Cumulative distribution of average transcript poly(A) tail length for NOT8*-expressing and GFP control cells. Figure 3—figure supplement 1—source data 1. Original files for western blot analysis displayed in . Figure 3—figure supplement 1—source data 2. File containing original western blots for , indicating the relevant bands and treatments.

Article Snippet: Antibody , Rabbit-anti XRN1 polyclonal , Thermo Fisher , A300-443A; RRID: AB_2219047 , (1:1000).

Techniques: Western Blot, Transfection, Control, Expressing

Journal: eLife

Article Title: Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress

doi: 10.7554/eLife.96284

Figure Lengend Snippet: ( a ) Nucleotide composition around the 5′ end of reads in arsenite-treated and unstressed cells upon XRN1 silencing. All reads were used, irrespective of adaptor ligation status. ( b, c ) Scatter plot of annotated 5′ ( a ) and 3ʹ ( b ) UTR length against transcript differential length in arsenite-treated and unstressed cells. The box plots on the right side summarize the y-axis variable for significant and nonsignificantly shortened transcripts. ( d, e ) Scatter plot of ribosome profiling footprint ( d ) and translational efficiency ( e ) levels against differential length in arsenite-treated and unstressed cells. Transcripts are stratified by significance of shortening. The Pearson’s correlation coefficient and the Mann–Whitney U test p-value are shown.

Article Snippet: Antibody , Rabbit-anti XRN1 polyclonal , Thermo Fisher , A300-443A; RRID: AB_2219047 , (1:1000).

Techniques: Ligation, MANN-WHITNEY

Journal: eLife

Article Title: Full-length direct RNA sequencing uncovers stress granule-dependent RNA decay upon cellular stress

doi: 10.7554/eLife.96284

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit-anti XRN1 polyclonal , Thermo Fisher , A300-443A; RRID: AB_2219047 , (1:1000).

Techniques: Control, Protease Inhibitor, Modification, Qubit Protein Assay, MTS Assay, Proliferation Assay, RNA Sequencing, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Membrane, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Sequencing, Software

Journal: bioRxiv

Article Title: Identification of transcriptome-wide cobalt chloride-induced hypoxia-responsive long noncoding RNAs regulated by cytoplasmic mRNA capping enzyme

doi: 10.1101/2024.11.21.624636

Figure Lengend Snippet: (A) Western blot analysis of U2OS cells expressing ± K294A showed Myc-tagged K294A expression. The quality of nuclear and cytoplasmic fractionation was validated by the detection of nuclear marker Lamin A/C and cytoplasmic marker GAPDH. (B) The steady-state expression of NEAT1-209, LUCAT1-211, and MSC-AS1-212 was significantly decreased in K294A-expressing cells. Statistical analysis was conducted using a two-tailed Student’s t-test. (C) The impact of siRNA-mediated Xrn1 knockdown in U2OS cells on the stability of the Xrn1 exoribonuclease was evaluated using Western blot analysis. Simultaneously, the Western blot for Myc verified the expression of the K294A variant. β-Actin served as the internal control. (D) The relative expression levels of Xrn1 protein, following transfection with Xrn1 siRNA, were quantified by normalization against β-Actin protein expression in ±K294A cells. (E) The steady-state levels of NEAT1-209, LUCAT1-211, and MSC-AS1-212 were significantly elevated following the transfection of siRNA Xrn1 into K294A U2OS cells. Statistical analysis was conducted using the two-way ANOVA. Values are expressed as mean ± standard deviation (SD) from three biological replicates. Statistical significance is denoted as follows: ns indicates non-significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001, with n ≥ 3.

Article Snippet: The XRN1 knockdown efficiency was measured by western blotting with rabbit anti-Xrn1 antibody (Thermo scientific cat no# PA5-57110).

Techniques: Western Blot, Expressing, Fractionation, Marker, Two Tailed Test, Knockdown, Variant Assay, Control, Transfection, Standard Deviation

Journal: Nature communications

Article Title: A rapid inducible RNA decay system reveals fast mRNA decay in P-bodies.

doi: 10.1038/s41467-024-46943-z

Figure Lengend Snippet: Fig. 4 | P-bodies are the site of fast RNA decay. ACT-MBS MEF cells were treated with siRNA against (a–c) XRN1, (d–f) DDX6, or (b–c, e–f) scrambled siRNA (NC) for 72 h. Cells were treated with Rapa and DRB, and fixed at different time points. smFISH-IF experiments were conducted with FISH probes against ACTB-MBS and mGAPDH, and antibodies against DCP1a. a Representative image at 2 h post induc- tion for XRN1 siRNA treated cells. The white box was enlarged on the right. ACTB- MBS FISH: magenta; mGAPDH FISH: cyan; DCP1a IF: green; DAPI: blue. Quantification of ACTB-MBS (b) or mGAPDH (c) mRNAs in P-bodies over 9 h time course after induction. XRN1 siRNA: diamonds; NC siRNA: circles. d Representative image for

Article Snippet: Samples were electrophoresed in a 4% SDS-PAGE gradient gel, transferred to a PVDF membrane, and blotted overnight with a 1:1000 dilution of rabbit anti-XRN1 primary antibody (Bethyl Laboratories A300-443A-M), 1:10000 dilution of rabbit anti-DDX6 primary antibody (Bethyl Laboratories A300-461A), or 1:250 rabbit antiRibosomal Protein S3 primary antibody (Santa Cruz sc-376008) as a control.

Techniques: