RNF4 Antibody [Unconjugated]

Western blot shows lysates of GM00719D human ataxia telangiectasia cell line either mock-treated (-) or exposed (+) to 10 Gy ionizing radiation (IR) and harvested after 1 hour, BaF3 mouse pro-B cell line, and LNCaP human prostate cancer cell line. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human RNF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7964) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for RNF4 at approximately 34 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

RNF4 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Goat Anti-Human RNF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7964) at 1.7 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to nuclei and cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

RNF4 was detected in immersion fixed paraffin-embedded sections of human testis using Goat Anti-Human RNF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7964) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to nuclei and cytoplasm. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Bortezomib causes elevated cell apoptosis in TKO cells. (a) Representative dose–response curve to bortezomib measured by CellTiter-Glo cell viability assay. Cell survival is normalized to the untreated control. The mean survival with SD of each concentration is shown. The asymmetrical (five-parameter) logistic dose–response model is used to fit the curve. (b) Bortezomib IC50 values. Each data point is an IC50 value from an experiment. Bars and error bars indicate the mean with SD. (c) Cell apoptosis after 48 h of bortezomib measured by Annexin V-PI staining. Each data point is a biological replicate and the mean with SD is shown. (d) Cartoon schematics of constructs used to complement TKO cells. EGFP, enhanced green fluorescent protein; SIM, SUMO-interacting motif; RING, really interesting new gene; TRAF, tumour necrosis factor receptor-associated factor; CD, catalytic domain; UBL, ubiquitin-like. (e) Western blot analyses of whole cell extracts of TKO cells expressing either EGFP-RNF4 or RNF4-EGFP. (f) Western blot analyses of whole cell extracts of TKO cells expressing WT USP7. (g) Western blot analyses of SUMOylated chromatin-bound proteins of TKO cells expressing either EGFP-RNF4 or RNF4-EGFP. Cells were treated with 10 µM of MG132 and 1 mM of HU for 4 h before collecting. (h) Cell apoptosis after 48 h of bortezomib measured by Annexin V-PI staining in TKO and complemented cells. (b,c,h) Each data point represents the average value from independent plates in an experiment. Bars and error bars indicate the mean and SD across multiple biological experiments. Significance was measured using an ordinary one-way ANOVA with Tukey's multiple comparisons test; n.s., not significant; **p ≤ 0.01; ***p ≤ 0.001. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37607592), licensed under a CC-BY license. Not internally tested by R&D Systems.

Global-genome repair of 5-azadC-induced DNMT1-DPCs monitored by PxP.a Schematic depiction of the experimental workflow used to monitor the repair of 5-azadC-induced DNMT1-DPCs. Cells were synchronized via a double thymidine block and released into early/mid S-phase for 3 h prior to induction of DNMT1-DPCs by a 30-min pulse of 5-azadC. Samples were collected either immediately after 5-azadC exposure or following a chase in drug-free media. Proteasome inhibitor (MG132, 5 µM), p97 inhibitor (p97i CB-5083, 5 µM) and SUMOylation inhibitor (SUMO-E1i ML-792, 5 µM) were added 1 h prior to induction of DPCs and kept during the chase with 5-azadC-free medium. Ubiquitylation inhibitor (Ub-E1i TAK-243, 1 µM) was added together with 5-azadC. b 5-azadC-induced DNMT1-DPC formation assessed by PxP. HeLa T-REx Flp-In cells were treated as depicted in (a) with the indicated doses of 5-azadC for 30 min prior to immediate isolation of DPCs using PxP and western blotting analysis. c–f 5-azadC-induced DNMT1-DPC formation and repair upon proteasome inhibition (c), inhibition of SUMOylation (d), inhibition of ubiquitylation (e), or knock-out of RNF4 (f) assessed by PxP. HeLa T-REx Flp-In cells were treated as depicted in (a) prior to extraction of DPCs using PxP, and analysis of samples by western blotting using the indicated antibodies. g HeLa WT and RNF4 knock-out (KO) cells were treated and analysed as depicted including an optional treatment with SUMOylation inhibitor (SUMO-E1i ML-792, 5 µM), prior to extraction of DPCs using PxP and analysis of samples by western blotting using the indicated antibodies. Experiments in (c–g) were repeated three times and similar results were obtained. Source data are provided as a Source Data file. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36681662), licensed under a CC-BY license. Not internally tested by R&D Systems.

Inhibition of SUMO‐targeted ubiquitylation partially restores UPS activity in thermally stressed G3BP1/2 knockout U2OS G3BP1/2 knockout expressing Ub‐YFP were transfected with control siRNA or RNF4 siRNA. Cell lysates were analyzed by immunoblot with RNF4 & Actin antibodies. Parental U2OS & G3BP1/2 KO expressing Ub‐YFP were transfected with control siRNA or RNF4 siRNA. Cell lysates were analyzed by immunoblot with RNF4 & Actin antibodies. Fluorescence micrographs of parental U2OS stably expressing Ub‐YFP, which had been transfected with control or RNF4 siRNA. were either left untreated (− heat shock), or exposed to 43°C for 30 min & followed for 4 h after heat shock (Recovery). Scale bar is 10 μm.Fluorescence micrographs of U2OS G3BP1/2 KO stably expressing Ub‐YFP, which had been transfected with control or RNF4 siRNA. were either left untreated (− heat shock), or exposed to 43°C for 30 min & followed for 4 h after heat shock (Recovery). Scale bar is 10 μm.Immunoblotting for PML in control (siControl) & PML‐depleted (siPML) parental & G3BP1/2 KO U2OS . Quantification of mean cellular YFP fluorescence intensities in U2OS expressing Ub‐YFP with & without PML siRNA transfection. The YFP fluorescence is normalized to untreated control . The frequency & distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, & dash lines represent the upper & lower interquartile range limits (n = 3 independent experiments, > 1,000 analyzed per condition, Kruskal‐Wallis test, *P 1,000 analyzed per condition, Kruskal‐Wallis test, *P 1,000 analyzed per condition, Kruskal‐Wallis test, *P 1,000 analyzed per condition, Kruskal‐Wallis test, *P < 0.05, **P < 0.01).Immunoblot of ataxin‐1 in parental & G3BP1/2 KO U2OS transfected with FLAGUb‐Ataxin‐1‐82Q.Immunoblot of FLAGUbiquitin in parental & G3BP1/2 KO U2OS transfected with FLAGUb‐Ataxin‐1‐82Q. Image collected & cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/36574355), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, IHC, ICC/IF
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated

RNF4 Antibody [Unconjugated] Summary

• Immunogen: E.coli-derived recombinant human RNF4
  Met1-Ile190
  Accession # P78317
• Specificity: Detects human RNF4 in direct ELISAs and Western blots.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 1-15 ug/mL
  • Immunohistochemistry 1-15 ug/mL
  • Western Blot 1 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for RNF4 Antibody [Unconjugated]

• E3 ubiquitin ligase RNF4
• EC 6.3.2.-
• Protein SNURF
• RES4-26
• ring finger protein 4SNURFSLX5
• RNF4
• SLX5
• Small nuclear ring finger protein
• SNURF

Background

RNF4 (small nuclear ring finger protein, SNURF) is a RING-finger ubiquitin E3 ligase that ubiquitinates and mediates the proteasomal destruction of targets such as PML, PEA3, CENP1, and PARP1. In addition to the RING domain, RNF4 contains four SUMO-interacting motifs (SIMs) that function to recruit this ligase to poly-sumoylated substrates. RNF4 will autoubiquitinate in vitro, and will also ubiquitinate poly-SUMO chains.

1. Geoffroy, M-C et al. (2010) Mol. Bio. Cell 21: 4227
2. Tathum, M.H. et al. (2008) Nat. Cell Bio. 10: 538

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for RNF4 Antibody (AF7964)(9)

Publications using AF7964

• Dürauer, S;Kang, HS;Wiebeler, C;Machida, Y;Schnapka, DS;Yaneva, D;Renz, C;Götz, MJ;Weickert, P;Major, AC;Rahmanto, AS;Gutenthaler-Tietze, SM;Daumann, LJ;Beli, P;Ulrich, HD;Sattler, M;Machida, YJ;Schwierz, N;Stingele, J; Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability Nature communications 2025-07-21 [PMID: 40691134] (Western Blot, Human)
• Tröster, V;Wong, RP;Börgel, A;Cakilkaya, B;Renz, C;Möckel, MM;Eifler-Olivi, K;Marinho, J;Reinberg, T;Furler, S;Schaefer, JV;Plückthun, A;Wolf, E;Ulrich, HD; Custom affinity probes reveal DNA-damage-induced, ssDNA-independent chromatin SUMOylation in budding yeast Cell reports 2025-02-26 [PMID: 40019834] (Western Blot, Human)
• Rodríguez Pérez, F;Natwick, D;Schiff, L;McSwiggen, D;Heckert, A;Huey, M;Morrison, H;Loo, M;Miranda, RG;Filbin, J;Ortega, J;Van Buren, K;Murnock, D;Tao, A;Butler, R;Cheng, K;Tarvestad, W;Zhang, Z;Gonzalez, E;Miller, RM;Kelly, M;Tang, Y;Ho, J;Anderson, D;Bashore, C;Basham, S; WRN inhibition leads to its chromatin-associated degradation via the PIAS4-RNF4-p97/VCP axis Nature communications 2024-07-18 [PMID: 39025847] (Western Blot, Human)
• Zhang, H;Xiong, Y;Sun, Y;Park, JM;Su, D;Feng, X;Keast, S;Tang, M;Huang, M;Wang, C;Srivastava, M;Yang, C;Zhu, D;Chen, Z;Li, S;Yin, L;Pommier, Y;Chen, J; RAD54L2-mediated DNA damage avoidance pathway specifically preserves genome integrity in response to topoisomerase 2 poisons Science advances 2023-12-08 [PMID: 38055811] (Western Blot, Human)
• Krawic, C;Luczak, MW;Valiente, S;Zhitkovich, A; Atypical genotoxicity of carcinogenic nickel(II): linkage to dNTP biosynthesis, DNA-incorporated rNMPs and impaired repair of TOP1-DNA crosslinks The Journal of biological chemistry 2023-10-25 [PMID: 37890780] (Western Blot, Human)
• Chang, YC;Lin, K;Baxley, RM;Durrett, W;Wang, L;Stojkova, O;Billmann, M;Ward, H;Myers, CL;Bielinsky, AK; RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication Open biology 2023-08-01 [PMID: 37607592] (Western Blot, Human)
• P Weickert, HY Li, MJ Götz, S Dürauer, D Yaneva, S Zhao, J Cordes, AC Acampora, I Forne, A Imhof, J Stingele SPRTN patient variants cause global-genome DNA-protein crosslink repair defects Nature Communications, 2023-01-21;14(1):352. 2023-01-21 [PMID: 36681662] (Western Blots, Human)
• MJ Young, YC Chen, SA Wang, HP Chang, WB Yang, CC Lee, CY Liu, YL Tseng, YC Wang, HS Sun, WC Chang, JJ Hung Estradiol-mediated inhibition of Sp1 decreases miR-3194-5p expression to enhance CD44 expression during lung cancer progression Journal of Biomedical Science, 2022-01-17;29(1):3. 2022-01-17 [PMID: 35034634] (Western Blot, Xenograft)
• Z Sha, T Blyszcz, R González-P, ACO Vertegaal, AL Goldberg Inhibiting ubiquitination causes an accumulation of SUMOylated newly synthesized nuclear proteins at PML bodies J. Biol. Chem., 2019-07-08;0(0):. 2019-07-08 [PMID: 31285264] (Western Blot, Human)

Bioinformatics

• Uniprot:
  • P78317(Human)