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Novus Biologicals rabbit anti rnf8
Rabbit Anti Rnf8, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+rnf8/10__3727_slash_096504018x15154085345907-86-19-22?v=Novus+Biologicals
Average 90 stars, based on 1 article reviews

rabbit anti rnf8 - by Bioz Stars, 2026-07

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Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: Key Resources Table

Article Snippet: Rabbit polyclonal anti-RNF8 , Proteintech , Cat#14112-1-AP.

Techniques: Recombinant, In Situ, Sequencing, Software

WRAP53β promotes recruitment of repair proteins to DSBs. ( A ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 24 h, exposed to IR (6 Gy) or left untreated, and, 1 h later, immunostained for γH2AX, MDC1, BRCA1, 53BP1, and RAD51. ( B ) U2OS cells treated as in A and then immunostained for RNF168 and conjugated ubiquitin (with the FK2 antibody). In the case of GFP-RNF8 staining, following treatment with oligonucleotides for 24 h, the cells were transiently transfected with the GFP-RNF8 plasmid for 8 h, exposed to IR (6 Gy), allowed to recover for 1 h, and then fixed and analyzed. ( C ) Quantification of the results in A and B as the percentage of 200 cells counted in each experiment whose nuclei contained IRIF. In the case of GFP-RNF8, only successfully transfected cells were counted. ( D ) U2OS cells were treated with the siRNAs indicated for 24 h, irradiated (6 Gy), allowed to recover for 1 h, and then subjected to Western blotting for WRAP53β, H2AX, and β-actin. The error bars depict the SEM. n = 3; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β promotes recruitment of repair proteins to DSBs. ( A ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 24 h, exposed to IR (6 Gy) or left untreated, and, 1 h later, immunostained for γH2AX, MDC1, BRCA1, 53BP1, and RAD51. ( B ) U2OS cells treated as in A and then immunostained for RNF168 and conjugated ubiquitin (with the FK2 antibody). In the case of GFP-RNF8 staining, following treatment with oligonucleotides for 24 h, the cells were transiently transfected with the GFP-RNF8 plasmid for 8 h, exposed to IR (6 Gy), allowed to recover for 1 h, and then fixed and analyzed. ( C ) Quantification of the results in A and B as the percentage of 200 cells counted in each experiment whose nuclei contained IRIF. In the case of GFP-RNF8, only successfully transfected cells were counted. ( D ) U2OS cells were treated with the siRNAs indicated for 24 h, irradiated (6 Gy), allowed to recover for 1 h, and then subjected to Western blotting for WRAP53β, H2AX, and β-actin. The error bars depict the SEM. n = 3; (**) P < 0.01; (***) P < 0.001, as determined by Student’s t -test.

Article Snippet: The following antibodies were used in immunoprecipitation, immunofluorescence, and Western blots: mouse α-γH2AX (Millipore, catalog no. 05-636), rabbit α-γH2AX (Cell Signaling, catalog no. 2577), rabbit α-H2AX (Abcam, catalog no. ab11175), rabbit α-MDC1 (Abcam, catalog no. ab11169), mouse α-MDC1 (Abcam, catalog no. ab50003), mouse α-pATM (Santa Cruz Biotechnology, catalog no. sc-47739), mouse α-RNF8 (Santa Cruz Biotechnology, catalog no. sc-271462), rabbit α-RNF8 (provided by Michael Huen), rabbit α-RNF168 (Millipore, catalog no. ABE367), mouse α-ubiquitin (FK2; Calbiochem, catalog no. ST1200), rabbit α-53BP1 (Novus Biologicals, catalog no. NB100-904), mouse α-BRCA1 (Santa Cruz Biotechnology, catalog no. sc-6954), rabbit α-RAD51 (Santa Cruz Biotechnology, catalog no. sc-8349), rabbit α-coilin (Santa Cruz Biotechnology, catalog no. sc-32860), mouse α-β-actin (Sigma), rabbit α-GFP (Abcam, catalog no. ab290), mouse α-Flag (Agilent Technologies, catalog no. 200472-21), normal rabbit IgG (Santa Cruz Biotechnology, catalog no. sc-2027), and normal mouse IgG (Santa Cruz Biotechnology, catalog no. sc-2025).

Techniques: Transfection, Staining, Plasmid Preparation, Irradiation, Western Blot

WRAP53β binds MDC1 and RNF8 via their FHA domains. ( A ) U2OS cells were either left untreated or irradiated with 6 Gy of IR, and, 30 min, later immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β, MDC1, GFP-RNF8, and β-actin. ( B ) U2OS cells were transfected with the indicated HA-MDC1 constructs for 16 h and irradiated with 2 Gy, and, 30 min later, immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β and HA-MDC1. ( C ) Schematic illustration of RNF8 deletion constructs. ( D ) U2OS cells were transiently transfected with EGFP-RNF8 plasmids, HA-MDC1, and Flag-WRAP53β for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for GFP-RNF8, Flag-WRAP53β, and HA-MDC1. (HC) Heavy chain of the antibody. U2OS ( E ) and H1299 ( F ) cells were transiently transfected with Flag-RNF8 plasmids, HA-MDC1, and EGFP-WRAP53β for 16 h; irradiated; and subjected to Flag immunoprecipitation followed by immunoblotting for the indicated proteins. ( G ) Schematic illustration of the domain architecture of MDC1 and RNF8, where black lines mark WRAP53β- and MDC1-binding sites. Numbers indicate amino acids.

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β binds MDC1 and RNF8 via their FHA domains. ( A ) U2OS cells were either left untreated or irradiated with 6 Gy of IR, and, 30 min, later immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β, MDC1, GFP-RNF8, and β-actin. ( B ) U2OS cells were transfected with the indicated HA-MDC1 constructs for 16 h and irradiated with 2 Gy, and, 30 min later, immunoprecipitation of WRAP53β was performed, followed by immunoblotting of WRAP53β and HA-MDC1. ( C ) Schematic illustration of RNF8 deletion constructs. ( D ) U2OS cells were transiently transfected with EGFP-RNF8 plasmids, HA-MDC1, and Flag-WRAP53β for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for GFP-RNF8, Flag-WRAP53β, and HA-MDC1. (HC) Heavy chain of the antibody. U2OS ( E ) and H1299 ( F ) cells were transiently transfected with Flag-RNF8 plasmids, HA-MDC1, and EGFP-WRAP53β for 16 h; irradiated; and subjected to Flag immunoprecipitation followed by immunoblotting for the indicated proteins. ( G ) Schematic illustration of the domain architecture of MDC1 and RNF8, where black lines mark WRAP53β- and MDC1-binding sites. Numbers indicate amino acids.

Techniques: Irradiation, Immunoprecipitation, Western Blot, Transfection, Construct, Binding Assay

WRAP53β facilitates MDC1–RNF8 interaction through its WD40 domain. ( A ) U2OS cells were treated with the siRNAs indicated for 48 h and with GFP-RNF8 for 24 h (all samples), irradiated with 6 Gy, and, 30-min later, subjected to immunoprecipitation of WRAP53β followed by immunoblotting of WRAP53β, MDC1, RNF8, γH2AX, and β-actin. ( B ) Immunoprecipitation of MDC1 in irradiated (6 Gy, 15-min recovery) U2OS cells treated with the siRNA indicated for 48 h or ATM inhibitor (ATMi) for 24 h. All samples were transfected with GFP-RNF8 for 16 h. ( C ) U2OS cells were treated with the siRNAs indicated for 48 h or ATM inhibitor for 16 h, irradiated with 6 Gy, allowed to recover for 15 min, and then subjected to Western blotting of MDC1, WRAP53β, γH2AX, and β-actin. ( D ) Schematic illustration of EGFP-tagged deletion constructs of WRAP53β. ( E ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids and Flag-RNF8 for 16 h, irradiated, and subjected to GFP immunoprecipitation followed by immunoblotting for MDC1, Flag-RNF8, and GFP-WRAP53β. (HC) Heavy chain of the antibody. ( F ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 8 h followed by transfection of EGFP-Empty or EGFP-WRAP53β WD40 (1–7) for 16 h, exposed to IR (6 Gy), and, after 1 h, immunostained for 53BP1 followed by quantification of the results. The graph in A shows the percentage of 100 GFP transfected cells in each experiment whose nuclei were 53BP1-positive. The error bars depict the SEM. n = 3; (*) P < 0.05, as determined by Student’s t -test. ( G ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids, HA-MDC1, and Flag-RNF8 for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for HA-MDC1, Flag-RNF8, and GFP-WRAP53β. ( H ) Schematic illustration of how WRAP53β scaffolds the MDC1–RNF8 complex. Upon DNA damage, WRAP53β binds MDC1 and RNF8 simultaneously via its WD40 domain and facilitates their interaction.

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: WRAP53β facilitates MDC1–RNF8 interaction through its WD40 domain. ( A ) U2OS cells were treated with the siRNAs indicated for 48 h and with GFP-RNF8 for 24 h (all samples), irradiated with 6 Gy, and, 30-min later, subjected to immunoprecipitation of WRAP53β followed by immunoblotting of WRAP53β, MDC1, RNF8, γH2AX, and β-actin. ( B ) Immunoprecipitation of MDC1 in irradiated (6 Gy, 15-min recovery) U2OS cells treated with the siRNA indicated for 48 h or ATM inhibitor (ATMi) for 24 h. All samples were transfected with GFP-RNF8 for 16 h. ( C ) U2OS cells were treated with the siRNAs indicated for 48 h or ATM inhibitor for 16 h, irradiated with 6 Gy, allowed to recover for 15 min, and then subjected to Western blotting of MDC1, WRAP53β, γH2AX, and β-actin. ( D ) Schematic illustration of EGFP-tagged deletion constructs of WRAP53β. ( E ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids and Flag-RNF8 for 16 h, irradiated, and subjected to GFP immunoprecipitation followed by immunoblotting for MDC1, Flag-RNF8, and GFP-WRAP53β. (HC) Heavy chain of the antibody. ( F ) U2OS cells were transfected with siControl or siWRAP53#2 oligonucleotides for 8 h followed by transfection of EGFP-Empty or EGFP-WRAP53β WD40 (1–7) for 16 h, exposed to IR (6 Gy), and, after 1 h, immunostained for 53BP1 followed by quantification of the results. The graph in A shows the percentage of 100 GFP transfected cells in each experiment whose nuclei were 53BP1-positive. The error bars depict the SEM. n = 3; (*) P < 0.05, as determined by Student’s t -test. ( G ) U2OS cells were transiently transfected with the indicated EGFP-WRAP53β plasmids, HA-MDC1, and Flag-RNF8 for 16 h; irradiated; and subjected to immunoprecipitation of GFP followed by immunoblotting for HA-MDC1, Flag-RNF8, and GFP-WRAP53β. ( H ) Schematic illustration of how WRAP53β scaffolds the MDC1–RNF8 complex. Upon DNA damage, WRAP53β binds MDC1 and RNF8 simultaneously via its WD40 domain and facilitates their interaction.

Techniques: Irradiation, Immunoprecipitation, Western Blot, Transfection, Construct

Schematic model of WRAP53β function in the DDR pathway. (Step 1) In response to IR, γH2AX and MDC1 accumulate at DSBs independently of WRAP53β. ATM-mediated phosphorylation of MDC1 makes MDC1 competent to bind RNF8. However, RNF8 is not yet localized at DSBs. (Step 2) WRAP53β is recruited to sites of DNA damage in an ATM-, H2AX-, and MDC1-dependent manner. Simultaneous binding of MDC1 and RNF8 to WRAP53β facilitates their direct interaction and retention of RNF8 at DSBs. (Step 3) Once assembled at DSBs, RNF8 catalyzes ubiquitylation of H2AX. Ubiquitylation at DSBs enables recruitment and accumulation of 53BP1, BRCA1, and RAD51 and subsequent DNA repair.

Journal: Genes & Development

Article Title: The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

doi: 10.1101/gad.246546.114

Figure Lengend Snippet: Schematic model of WRAP53β function in the DDR pathway. (Step 1) In response to IR, γH2AX and MDC1 accumulate at DSBs independently of WRAP53β. ATM-mediated phosphorylation of MDC1 makes MDC1 competent to bind RNF8. However, RNF8 is not yet localized at DSBs. (Step 2) WRAP53β is recruited to sites of DNA damage in an ATM-, H2AX-, and MDC1-dependent manner. Simultaneous binding of MDC1 and RNF8 to WRAP53β facilitates their direct interaction and retention of RNF8 at DSBs. (Step 3) Once assembled at DSBs, RNF8 catalyzes ubiquitylation of H2AX. Ubiquitylation at DSBs enables recruitment and accumulation of 53BP1, BRCA1, and RAD51 and subsequent DNA repair.

Techniques: Binding Assay

Figure 1. Mono-ubiquitination of PCNA by RNF8. (A) Mono-ubiquitination of PCNA by RNF8 and UbcH5c. The ubiquitination of PCNA was assayed in a reconstituted system using purified human proteins (Experimental Procedures). The reaction mixtures contained 10 μg of ubiquitin, 30 nM E1 ubiquitin acti- vating enzyme, 500 nM of UbcH5c, 100 ng of PCNA and 100 ng of RNF8. After incubation at 30°C for the indicated times, the reaction products were examined by SDS-PAGE and Western blotting with anti-PCNA (Experimental Procedures). Positions of PCNA and mono-ubiquitinated PCNA are shown by the arrowheads. (B) Confirmation of the identity of mono-ubiquitinated PCNA. The blot shown in (A) was stripped and re-blotted with anti-ubiquitin. (The high molecular weight products are polyubiquitinated products that are expected from the auto-polyubiquitination of RNF8).

Journal: Cell cycle (Georgetown, Tex.)

Article Title: PCNA is ubiquitinated by RNF8.

doi: 10.4161/cc.7.21.6949

Figure Lengend Snippet: Figure 1. Mono-ubiquitination of PCNA by RNF8. (A) Mono-ubiquitination of PCNA by RNF8 and UbcH5c. The ubiquitination of PCNA was assayed in a reconstituted system using purified human proteins (Experimental Procedures). The reaction mixtures contained 10 μg of ubiquitin, 30 nM E1 ubiquitin acti- vating enzyme, 500 nM of UbcH5c, 100 ng of PCNA and 100 ng of RNF8. After incubation at 30°C for the indicated times, the reaction products were examined by SDS-PAGE and Western blotting with anti-PCNA (Experimental Procedures). Positions of PCNA and mono-ubiquitinated PCNA are shown by the arrowheads. (B) Confirmation of the identity of mono-ubiquitinated PCNA. The blot shown in (A) was stripped and re-blotted with anti-ubiquitin. (The high molecular weight products are polyubiquitinated products that are expected from the auto-polyubiquitination of RNF8).

Article Snippet: The knockdown efficiency was evaluated by Western blotting with rabbit polyclonal antibody against anti-RNF8 (Abcam Inc., Cambridge, MA).

Techniques: Ubiquitin Proteomics, Purification, Incubation, SDS Page, Western Blot, High Molecular Weight

Figure 2. Dependence of the Mono-Ubiquitination Reaction on PCNA and RNF8 Concentrations. (A) RNF8 concentration dependence. The reactions were performed as in (A) with increasing concentrations of RNF8 from 1 to 300 ng/assay for 1 hr. “C” refers to a control reaction in which ATP was omitted in this and subsequent panels. (B) PCNA concentration dependence. The reactions were performed as in (A) with increasing concentrations of PCNA from 5 to 120 ng/assay for 1 hr. “C” refers to the control reaction in which ATP was omitted. (C) RNF8 interacts with PCNA. The ability of PCNA to interact with RNF8 was examined by overlay blotting with digoxigenin- PCNA as previously described.40 Lane M, protein molecular weight markers; lane 1, glutathione-S-transferase (0.5 μg); lane 2, his-tagged RNF8 (0.5 μg). The specificity of the interaction is supported by the failure to observe signals for the molecular weight markers and for glutathione-S-transferase. (D) Rad6, Ubc13/Uev1a, UbcH6, UbcH2 and UbcH3 do not function as E2 enzymes with RNF8 for the mono-ubiquitination of PCNA. PCNA was incubated with RNF8 and either UbcH5c (lane1), Rad6 (lane2), Ubc13/ Uev1a (lane 3), UbcH6 (lane 4), UbcH2 (lane5) or UbcH3 (lane 6), as described in Experimental Procedures. Products were analyzed by Western blotting with anti-PCNA.

Journal: Cell cycle (Georgetown, Tex.)

Article Title: PCNA is ubiquitinated by RNF8.

doi: 10.4161/cc.7.21.6949

Figure Lengend Snippet: Figure 2. Dependence of the Mono-Ubiquitination Reaction on PCNA and RNF8 Concentrations. (A) RNF8 concentration dependence. The reactions were performed as in (A) with increasing concentrations of RNF8 from 1 to 300 ng/assay for 1 hr. “C” refers to a control reaction in which ATP was omitted in this and subsequent panels. (B) PCNA concentration dependence. The reactions were performed as in (A) with increasing concentrations of PCNA from 5 to 120 ng/assay for 1 hr. “C” refers to the control reaction in which ATP was omitted. (C) RNF8 interacts with PCNA. The ability of PCNA to interact with RNF8 was examined by overlay blotting with digoxigenin- PCNA as previously described.40 Lane M, protein molecular weight markers; lane 1, glutathione-S-transferase (0.5 μg); lane 2, his-tagged RNF8 (0.5 μg). The specificity of the interaction is supported by the failure to observe signals for the molecular weight markers and for glutathione-S-transferase. (D) Rad6, Ubc13/Uev1a, UbcH6, UbcH2 and UbcH3 do not function as E2 enzymes with RNF8 for the mono-ubiquitination of PCNA. PCNA was incubated with RNF8 and either UbcH5c (lane1), Rad6 (lane2), Ubc13/ Uev1a (lane 3), UbcH6 (lane 4), UbcH2 (lane5) or UbcH3 (lane 6), as described in Experimental Procedures. Products were analyzed by Western blotting with anti-PCNA.

Article Snippet: The knockdown efficiency was evaluated by Western blotting with rabbit polyclonal antibody against anti-RNF8 (Abcam Inc., Cambridge, MA).

Techniques: Ubiquitin Proteomics, Concentration Assay, Control, Molecular Weight, Incubation, Western Blot

Figure 3. Polyubiquitination of PCNA by RNF8. (A) PCNA is polyubiquit- inated by RNF8-Ubc13/Uev1a after its prior monoubiquitination by RNF8- UbcH5c. PCNA was incubated with RNF8 and UbcH5c, or with RNF8, UbcH5c and Ubc13/Uev1a for periods of 1, 2 and 3 h and analyzed for formation of ubiquitinated PCNA. “C” refers to a control reaction in which ATP was omitted. Positions of the protein molecular weight markers (kDa) are shown on the right for all panels. (B) PCNA is ubiquitinated on K164. The ubiquitination of PCNA and its K164R mutant by RNF8 were assayed as described in Experimental Procedures. His-PCNA and his-PCNA-K164R were used for this experiment. Ubiquitination by RNF8-UbcH5c: Lane 1, his- PCNA with ATP omitted; lanes 2 and 4 (duplicates), his-PCNA-K164R; lane 3, his-PCNA. Ubiquitination by RNF8, UbcH5c and Ubc13/Uev1a: lane 5, his-PCNA; lane 6, his-PCNA-K164R. Ubiquitination by RNF8-Ubc13/ Uev1a: lane 7, his-PCNA; lane 8, his-PCNA-K164R. (C) Polyubiquitination of PCNA occurs through K63 isopeptide linkages. PCNA was ubiquitinated using ubiquitin and its K64R, K48R, K48R/K64R or K0 mutants. Assays were performed with RNF8 and UbcH5c (lanes 1–3) or with RNF8, UbcH5c and Ubc13/Uev1a (lanes 4–7). [The greater amount of polyubiquitinated PCNA observed with the ubi-K48R mutant (lane 5) compared to ubiquitin (lane 4) is likely due to the fact that the RNF8 auto-ubiquitination reaction is suppressed, i.e., competition for the ubiquitin substrate is reduced].

Journal: Cell cycle (Georgetown, Tex.)

Article Title: PCNA is ubiquitinated by RNF8.

doi: 10.4161/cc.7.21.6949

Figure Lengend Snippet: Figure 3. Polyubiquitination of PCNA by RNF8. (A) PCNA is polyubiquit- inated by RNF8-Ubc13/Uev1a after its prior monoubiquitination by RNF8- UbcH5c. PCNA was incubated with RNF8 and UbcH5c, or with RNF8, UbcH5c and Ubc13/Uev1a for periods of 1, 2 and 3 h and analyzed for formation of ubiquitinated PCNA. “C” refers to a control reaction in which ATP was omitted. Positions of the protein molecular weight markers (kDa) are shown on the right for all panels. (B) PCNA is ubiquitinated on K164. The ubiquitination of PCNA and its K164R mutant by RNF8 were assayed as described in Experimental Procedures. His-PCNA and his-PCNA-K164R were used for this experiment. Ubiquitination by RNF8-UbcH5c: Lane 1, his- PCNA with ATP omitted; lanes 2 and 4 (duplicates), his-PCNA-K164R; lane 3, his-PCNA. Ubiquitination by RNF8, UbcH5c and Ubc13/Uev1a: lane 5, his-PCNA; lane 6, his-PCNA-K164R. Ubiquitination by RNF8-Ubc13/ Uev1a: lane 7, his-PCNA; lane 8, his-PCNA-K164R. (C) Polyubiquitination of PCNA occurs through K63 isopeptide linkages. PCNA was ubiquitinated using ubiquitin and its K64R, K48R, K48R/K64R or K0 mutants. Assays were performed with RNF8 and UbcH5c (lanes 1–3) or with RNF8, UbcH5c and Ubc13/Uev1a (lanes 4–7). [The greater amount of polyubiquitinated PCNA observed with the ubi-K48R mutant (lane 5) compared to ubiquitin (lane 4) is likely due to the fact that the RNF8 auto-ubiquitination reaction is suppressed, i.e., competition for the ubiquitin substrate is reduced].

Article Snippet: The knockdown efficiency was evaluated by Western blotting with rabbit polyclonal antibody against anti-RNF8 (Abcam Inc., Cambridge, MA).

Techniques: Incubation, Control, Molecular Weight, Ubiquitin Proteomics, Mutagenesis

Figure 4. RNF8 is required for the Mono-ubiquitination of PCNA that is Induced by DNA Damage Treatments with UV or MNNG. (A) Suppression of RNF8 expression by RNAi interference. A549 cells were transfected with vectors for four 29 mer shRNAs (Experimental Procedures). RNF8 levels were examined by Western blotting with anti-RNF8 for the seven clonal cell populations that were used in this experiment. These were R3-12, R4-9, R4-10, R2-6, R4-2, R4-3 and R4-4, where the prefixes R2, R3 and R4 refer to the shRNA used, and the suffixes refer to the isolates. “RC” refers to A549 cells that were transfected with control shRNA. (B) RNF8 knockdown suppresses mono-ubiquitination of PCNA after UV treatment. R3-12, R4-9, R4-10 and R2-6 cells were treated with UV (20 J/m2) and harvested for analysis four hours later and Western blotted for PCNA. “RC”, cells transfected with control shRNA. “C”, parent A549 cells that were not transfected. (C) RNF8 knockdown suppresses mono-ubiquitination of PCNA after MNNG treatment. The R4-2, R4-3 and R4-4 cell lines were treated with 100 ng/ml MNNG for 4 h, and Western blotted for PCNA (lanes 2–4). A parallel experiment was performed where the cells were UV-treated as in (B) (lanes 6–8). The positions of PCNA and mono-ubiquitinated PCNA are indicated by arrowheads.

Journal: Cell cycle (Georgetown, Tex.)

Article Title: PCNA is ubiquitinated by RNF8.

doi: 10.4161/cc.7.21.6949

Figure Lengend Snippet: Figure 4. RNF8 is required for the Mono-ubiquitination of PCNA that is Induced by DNA Damage Treatments with UV or MNNG. (A) Suppression of RNF8 expression by RNAi interference. A549 cells were transfected with vectors for four 29 mer shRNAs (Experimental Procedures). RNF8 levels were examined by Western blotting with anti-RNF8 for the seven clonal cell populations that were used in this experiment. These were R3-12, R4-9, R4-10, R2-6, R4-2, R4-3 and R4-4, where the prefixes R2, R3 and R4 refer to the shRNA used, and the suffixes refer to the isolates. “RC” refers to A549 cells that were transfected with control shRNA. (B) RNF8 knockdown suppresses mono-ubiquitination of PCNA after UV treatment. R3-12, R4-9, R4-10 and R2-6 cells were treated with UV (20 J/m2) and harvested for analysis four hours later and Western blotted for PCNA. “RC”, cells transfected with control shRNA. “C”, parent A549 cells that were not transfected. (C) RNF8 knockdown suppresses mono-ubiquitination of PCNA after MNNG treatment. The R4-2, R4-3 and R4-4 cell lines were treated with 100 ng/ml MNNG for 4 h, and Western blotted for PCNA (lanes 2–4). A parallel experiment was performed where the cells were UV-treated as in (B) (lanes 6–8). The positions of PCNA and mono-ubiquitinated PCNA are indicated by arrowheads.

Article Snippet: The knockdown efficiency was evaluated by Western blotting with rabbit polyclonal antibody against anti-RNF8 (Abcam Inc., Cambridge, MA).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Western Blot, shRNA, Control, Knockdown

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