rabbit polyclonal anti cpsf3 antibody  (Bethyl)

Journal: Cell reports

Article Title: Nuclear eIF4E Stimulates 3′-End Cleavage of Target RNAs

doi: 10.1016/j.celrep.2019.04.008

Figure Lengend Snippet: (A) Endogenous eIF4E immunoprecipitated (IP) with CPSF1 and CPSF3 in nuclear lysates from U2OS cells. Fractionation controls for nuclear IP were Lamin and RNA polymerase II for the nucleus (N) and GAPDH and tubulin for the cytoplasm (C). Nc, nuclear lysate; S, supernatant. (B and D) Diagram of constructs, which is not to scale, with the position of primers to detect cleaved (reverse primer immediately downstream the cleavage site (C/A) + 13 thymine nucleotides, shown in pink (qRT-PCR; ) and uncleaved LacZ transcripts (primers in red, forward used for both cleaved and uncleaved) (B). (D) depicts constructs used for studies into the deletion of the PAS region. (C) eIF4E-FLAG RIP using primers that bracket the cleavage site as used for the cleavage assay (uncleaved primers [red], exon “total” primers [green]). Results are relative to IgG controls. These findings indicate that, in the nucleus, eIF4E associated with uncleaved LacZ-4ESE RNA. (E) eIF4E-FLAG RIP from nuclear lysates shows PAS enriched association with LacZ-4ESE by 2-fold relative to LacZ-4ESEΔPAS. As expected, eIF4E did not interact with VEGF RNA in nuclear lysates. Experiments were three biological replicates each performed in technical duplicates for (C) and (E) and three biological replicates for (A), each performed in technical triplicates. For qRT-PCR experiments, values are means ± SD with p values as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Rabbit polyclonal anti- CPSF3 antibody , Bethyl , Cat # A301–090A; RRID:AB_873009.

Techniques: Immunoprecipitation, Fractionation, Construct, Quantitative RT-PCR, Cleavage Assay

Journal: Cell reports

Article Title: Nuclear eIF4E Stimulates 3′-End Cleavage of Target RNAs

doi: 10.1016/j.celrep.2019.04.008

Figure Lengend Snippet: (A) Endogenous CPSF3 RIPs ACTIN , LacZ , and LacZ-4ESE RNAs relative to IgG controls in nuclear lysates from eIF4E-FLAG U2OS cells. (B) eIF4E-FLAG RIP as a function of RNA knockdown to CPSF3 (si CPSF3 ) relative to RNAi controls (siCtrl) showed reduced binding of eIF4E with LacZ-4ESE RNAs compared with siCtrls. RIPs were relative to IgG controls, and Lamin is a negative control. (C) si CPSF3 increased levels of uncleaved RNA (i.e., reduced cleavage) for both LacZ-4ESE and LacZ RNAs relative to siCtrl by qRT-PCR in nuclear lysates from eIF4E-FLAG U2OS cells. (D) si CPSF3 impaired nuclear export of all RNAs examined. The cytoplasmic-to-nuclear RNA ratio (C/N ratio) is shown. (A), (C), and (D) were repeated in three biological replicates and (B) in two biological replicates, all with technical triplicates. Values are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Student’s t test was used for calculation of p values for for (A)–(C), whereas ANOVA was used for (D). Knockdown controls and total RNA levels are in .

Article Snippet: Rabbit polyclonal anti- CPSF3 antibody , Bethyl , Cat # A301–090A; RRID:AB_873009.

Techniques: Knockdown, Binding Assay, Negative Control, Quantitative RT-PCR

Journal: Cell reports

Article Title: Nuclear eIF4E Stimulates 3′-End Cleavage of Target RNAs

doi: 10.1016/j.celrep.2019.04.008

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: Rabbit polyclonal anti- CPSF3 antibody , Bethyl , Cat # A301–090A; RRID:AB_873009.

Techniques: Software, Recombinant