nucleolin polyclonal antibody  (Proteintech)

Journal: bioRxiv

Article Title: Dependency Map correlation analysis reveals WDR89 as a genome maintenance factor

doi: 10.64898/2026.01.28.698016

Figure Lengend Snippet: WDR89 is a nucleolar protein that responds to double-strand DNA break-inducing stresses. ATM-dependent nucleolar accumulation of WDR89 upon genotoxic stress. (A) WDR89 localizes predominantly in the nucleus and overlaps largely with nucleolin. (B) Quantification of WDR89 nuclear staining intensity following ionizing radiation. (C) Quantification of WDR89 nuclear staining intensity following genotoxic chemical treatments. (D) Confirmation of DNA damage by γH2AX foci staining of the conditions in C. (E) Effect of ATM inhibitor treatment on WDR89 nuclear intensity changes after IR. (F) Western blot of chromatin fractionation for WDR89 and the indicated markers. For panels in B, C, D and E, ****p<0.0001, by ANOVA with Tukey’s post-hoc test or by T-test where appropriate.

Article Snippet: Immunofluorescence staining was performed using anti-γ-H2AX antibodies (Abcam, ab81299, Santa Cruz, sc-517348), an anti-Nucleolin antibody (Novus, NBP2-44610), anti WDR89 antibody (Bethyl, A301-872A) or p53 antibody (Cell Signaling, #9282) at 1:250 dilution and Alexafluor 568 goat anti-rabbit secondary and Alexafluor 488 goat anti-mouse antibodies (Invitrogen) at 1:400 dilution.

Techniques: Staining, Western Blot, Fractionation

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: Identification of PAR-binding proteins using an RBDs array. ( A ) Structural similarities between RNA and PAR. ( B ) Several poly(A)-binding proteins were identified using the RBDs array probed with biotin-poly(A) (8-mer). ( C ) Anti-GST antibody serves as a loading control for panel ( B ). ( D, E ) Three proteins (PABPN1, PABPN1L, and NCL) were identified as PAR-binding on the RBDs array. Arrays were probed with biotin-PAR5 (5-mer) ( D ) and biotin-PAR20 (20-mer) ( E ). ( F ) The anti-GST antibody array serves as a loading control for both panels ( D ) and ( E ). ( G ) The poly(A) and PAR binders are marked on the array and listed on the right. Biotinylated probes were labeled with Cy3-streptavidin.

Article Snippet: The following antibodies were used for immunoblotting: Flag (Sigma–Aldrich, Cat #F3165), GFP (Invitrogen, Cat #A6455), PAR (CST, Cat #89190S), and NCL (CST, Cat #14574S).

Techniques: Binding Assay, Control, Ab Array, Labeling

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: Comparative interactions on a focused PAR-binding array. ( A–C ) A focused protein domain array containing previously reported PAR-binding domains and the RRMs of PABPN1 and NCL was used to validate binding with biotinylated PAR5 ( A ), PAR20 ( B ), and PAR-long ( C ). ( D ) Anti-GST antibody serves as a loading control for panels ( A )–( C ). ( E ) Map and ( F ) key of the arrayed proteins shown in panels ( A )–( D ). The array includes newly identified PAR binders from Fig. , as well as previously known PAR-binding domains, including Macro, PBM, PBZ, WWE, OB Fold, PIN, RRM, and BRCT domains. In the key ( F ), green-highlighted domains indicate positive PAR binders (both strong and weak). ( G ) Pull-down assay validates the interaction between PAR-long and the RRMs of PABPN1 and NCL. ( H ) Interaction mapping between PAR chains and individual RRM domains of NCL. ( I ) Map and ( J ) key of the arrayed proteins shown in panel ( H ). In the key ( J ), green-highlighted domains indicate interactions with PAR chains. ( K ) Pull-down assay validates the interaction between PAR-long and NCL RRM [ – ]. Biotinylated probes were labeled with Cy3-streptavidin. In maps [panels ( E ) and ( I )], “c” represents GST alone. Gray dots represent empty spaces.

Article Snippet: The following antibodies were used for immunoblotting: Flag (Sigma–Aldrich, Cat #F3165), GFP (Invitrogen, Cat #A6455), PAR (CST, Cat #89190S), and NCL (CST, Cat #14574S).

Techniques: Binding Assay, Control, Pull Down Assay, Labeling

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: PARylation-dependent recruitment of NCL and PABPN1 to DNA damage sites. ( A ) Live-cell imaging snapshots showing ectopic GFP-NCL and GFP-PABPN1 recruitment to laser-induced DNA damage sites in U2OS cells. Recruitment was blocked by PARP1 inhibition with PJ34. Endogenous PARP1 was detected using an RFP-fused nanobody. ( B ) Binding of NCL to PARP1 in U2OS cells was promoted by H 2 O 2 and H 2 O 2 /PARGi treatments. DNase and RNase treatment did not impact the NCL/PARP1 interaction. An anti-Flag IP was performed on lysates from Flag-PARP1 overexpressed cells, followed by immunoblotting with Flag and NCL antibodies. ( C ) Binding of PABPN1 to PARP1 in U2OS cells was promoted by H 2 O 2 and H 2 O 2 /PARGi treatment. DNase and RNase treatment did not impact the PABPN1/PARP1 interaction. An anti-Flag IP was performed on lysates from Flag-PARP1 and GFP-PABPN1 co-expressed cells, followed by immunoblotting with Flag and GFP antibodies.

Article Snippet: The following antibodies were used for immunoblotting: Flag (Sigma–Aldrich, Cat #F3165), GFP (Invitrogen, Cat #A6455), PAR (CST, Cat #89190S), and NCL (CST, Cat #14574S).

Techniques: Live Cell Imaging, Inhibition, Binding Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: The RRM of PABPN1 binds PAR and RNA competitively. ( A, B ) Pull-down analysis shows that the mutation of two aromatic residues (mR) in the RRM of PABPN1 blocks poly(A) RNA binding ( A ) but not PAR-long chain binding ( B ). ( C ) Live-cell imaging snapshots show the recruitment of ectopic GFP-PABPN1 and mutant GFP-PABPN1-mR to laser-induced DNA damage sites in U2OS cells. Recruitment was blocked by the PARP1 inhibitor PJ34. ( D ) Kinetic characterization of RNA and PAR binding to the RRMs of PABPN1 and NCL utilizing single cycle SPR. ( E ) FP assays showed that PABPN1 RRM binding to PAR and RNA is competitive in vitro . GST-RRM of PABPN1 was either incubated with poly(A) and titrated with increasing concentrations of PAR5 (PAR5 competition; black trace) or incubated with PAR5 and titrated with increasing concentrations of poly(A) (RNA competition; blue trace). ( F, G ) Pull-down assays demonstrate that PABPN1 RRM binding to PAR and RNA is competitive in vitro . GST-RRM of PABPN1 was either incubated with biotinylated PAR-long overnight, followed by incubation with varying concentrations of poly(A) for 2 h ( F ), or incubated with biotinylated poly(A) overnight, followed by incubation with varying concentrations of PAR-long for 2 h ( G ). Samples were analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blotting with an anti-GST antibody. Poly(A) 8-mer RNA was used in all experiments.

Article Snippet: The following antibodies were used for immunoblotting: Flag (Sigma–Aldrich, Cat #F3165), GFP (Invitrogen, Cat #A6455), PAR (CST, Cat #89190S), and NCL (CST, Cat #14574S).

Techniques: Mutagenesis, RNA Binding Assay, Binding Assay, Live Cell Imaging, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot