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H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay ( a ) and the fork degradation assay ( b ). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or <t>REV7</t> siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a file.
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H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay ( a ) and the fork degradation assay ( b ). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or <t>REV7</t> siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a file.
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H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay ( a ) and the fork degradation assay ( b ). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or <t>REV7</t> siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a file.
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H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay ( a ) and the fork degradation assay ( b ). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or <t>REV7</t> siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a file.
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H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay ( a ) and the fork degradation assay ( b ). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or <t>REV7</t> siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a file.
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H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay ( a ) and the fork degradation assay ( b ). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or REV7 siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a file.

Journal: Nature Communications

Article Title: The RING finger E3 ligase RNF25 protects DNA replication forks independently of its canonical roles in ubiquitin signaling

doi: 10.1038/s41467-025-62368-8

Figure Lengend Snippet: H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay ( a ) and the fork degradation assay ( b ). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or REV7 siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log 2 -transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a file.

Article Snippet: Antibodies used: TRIM11 (ab111694), pIRF3 S386 (ab76493), MUS81 (ab14387), and MRE11 (ab214) from Abcam; RNF25 (A303-844A), pRPA S33 (A300-246A), pRPA S4/S8 (A300-245A), HLTF (A300-230A), Pol η (A301-231A), Pol κ (A301-975A), Pol ι (A301-304A), RAD18 (A301-340A), and HA (A190-138A) from Bethyl Laboratories; Vinculin (V4505), SHLD3 (HPA068764), and γH2AX (05-636) from Sigma Millipore; RPA 34 (NA19L) from Calbiochem; pATM S1981 (sc-47739), β-actin (sc-47778), GAPDH (sc-32233), TNKS1/2 (sc-365897), PARP1 (sc-8007), UBE2D2 (sc-100617), REV1 (sc-393022), FANCD2 (sc-28194), Histone H2B (sc-10808), WEE1 (sc-9037), CDK2 pT14/Y15 (sc-28435-R), CDK2 (sc-6248), and PCNA (sc-56) from Santa Cruz Biotech; CHK1 (CST 2360), CHK1 pS317 (CST 2344), CDC2 pTyr15 (CST 9111), PIAS1 (CST 3550), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST 9106), pSTING Ser366 (CST 19781), STING (CST 13647), pSTAT1 Tyr701 (CST 9167) and STAT1 (CST 9175) from Cell Signaling Technology; REV7 (12683-1-AP), EXO1 (16253-1-AP) from Proteintech; CtIP (61141) from Active Motif.

Techniques: Transfection, Motility Assay, Degradation Assay, Two Tailed Test, MANN-WHITNEY, Immunoprecipitation, Control, Transformation Assay, Expressing, Mutagenesis, Homologous Recombination, Amplification

H1299 WT and RNF25 KO cells were transfected with indicated siRNAs for 48 h before conducting the DNA fiber fork degradation ( a ) or fork movement ( b ) assay. 150 fibers were analyzed per condition with means shown using bars. This Fig. is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c H1299 cells were transfected with indicated siRNAs before HA-RNF25 was expressed using adenoviral vectors and immunoprecipitated with anti-HA beads. d Schematic depicting the role of RNF25 in basal replication and following replication stress. Under basal conditions, RNF25 promotes fork movement along with REV7 and REV3L. Under replication stress conditions, RNF25 protects reversed forks by recruiting REV7. REV1 and REV3L also function in the same fork protection pathway. However, in RNF25 deficient cells, fork degradation is mediated by nucleases MRE11 and CtIP. Source data are provided as a file.

Journal: Nature Communications

Article Title: The RING finger E3 ligase RNF25 protects DNA replication forks independently of its canonical roles in ubiquitin signaling

doi: 10.1038/s41467-025-62368-8

Figure Lengend Snippet: H1299 WT and RNF25 KO cells were transfected with indicated siRNAs for 48 h before conducting the DNA fiber fork degradation ( a ) or fork movement ( b ) assay. 150 fibers were analyzed per condition with means shown using bars. This Fig. is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c H1299 cells were transfected with indicated siRNAs before HA-RNF25 was expressed using adenoviral vectors and immunoprecipitated with anti-HA beads. d Schematic depicting the role of RNF25 in basal replication and following replication stress. Under basal conditions, RNF25 promotes fork movement along with REV7 and REV3L. Under replication stress conditions, RNF25 protects reversed forks by recruiting REV7. REV1 and REV3L also function in the same fork protection pathway. However, in RNF25 deficient cells, fork degradation is mediated by nucleases MRE11 and CtIP. Source data are provided as a file.

Article Snippet: Antibodies used: TRIM11 (ab111694), pIRF3 S386 (ab76493), MUS81 (ab14387), and MRE11 (ab214) from Abcam; RNF25 (A303-844A), pRPA S33 (A300-246A), pRPA S4/S8 (A300-245A), HLTF (A300-230A), Pol η (A301-231A), Pol κ (A301-975A), Pol ι (A301-304A), RAD18 (A301-340A), and HA (A190-138A) from Bethyl Laboratories; Vinculin (V4505), SHLD3 (HPA068764), and γH2AX (05-636) from Sigma Millipore; RPA 34 (NA19L) from Calbiochem; pATM S1981 (sc-47739), β-actin (sc-47778), GAPDH (sc-32233), TNKS1/2 (sc-365897), PARP1 (sc-8007), UBE2D2 (sc-100617), REV1 (sc-393022), FANCD2 (sc-28194), Histone H2B (sc-10808), WEE1 (sc-9037), CDK2 pT14/Y15 (sc-28435-R), CDK2 (sc-6248), and PCNA (sc-56) from Santa Cruz Biotech; CHK1 (CST 2360), CHK1 pS317 (CST 2344), CDC2 pTyr15 (CST 9111), PIAS1 (CST 3550), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST 9106), pSTING Ser366 (CST 19781), STING (CST 13647), pSTAT1 Tyr701 (CST 9167) and STAT1 (CST 9175) from Cell Signaling Technology; REV7 (12683-1-AP), EXO1 (16253-1-AP) from Proteintech; CtIP (61141) from Active Motif.

Techniques: Transfection, Two Tailed Test, MANN-WHITNEY, Immunoprecipitation