rabbit rtf2  (Proteintech)

Journal: Molecular cell

Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

doi: 10.1016/j.molcel.2017.11.035

Figure Lengend Snippet: (A) Schematic of SILAC/iPOND experiment comparing replication fork occupancy during aphidicolin treatment in control vs. DDI1/2 depleted cells. The experiment was performed twice, with light/heavy label swap. (B) Graph of the Log(2) ratio of a curated list of DDI-target candidates among replication and repair proteins identified by iPOND in DDI1/2 co-depleted/control cells in two label swap experiments. Proteins reaching the enrichment threshold of Log(2)=1/−1 are named and shown in red. Proteins were quantified using the average area. (C) Validation of mass spectrometry results by iPOND and western blot. Cells were treated with 0.4 μM aphidicolin for 3 h then labeled with EdU for 50′ in the presence of aphidicolin. For chase, cells were not treated with aphidicolin and were labeled with EdU for 10′ then with thymidine for 50′. (D) Western blot showing iPOND analysis of H2B, MCM7, and RTF2 during unperturbed replication. U2OS cells were labeled with EdU for 10′ followed by 0′, 30′, or 60′ of thymidine chase. (E) Validation of RTF2 stabilization in whole cell extract. Control or DDI1/2 co-depleted cells were treated with the indicated siRNAs against RTF2 or control. Error bars represent SEM n=at least 3. (F) Quantification of relative protein levels of RTF2 in control or DDI1/2 depleted cells. (G) Quantification of relative mRNA levels of RTF2 in control, RTF2, or DDI1/2 depleted cells. (H) Coimmunoprecipitation of GFP-DDI2 and RTF2. Stably expressing GFP-DDI2 U2OS cells treated with 2 μM MG132 were subject to immunoprecipitation by anti-GFP or an IgG control, using an IP buffer containing 5 mg/ml NEM, and immunoblotted for RTF2. (I) Graph showing survival of U2OS cells treated with the indicated doses of HU. Cells were depleted of MRE11, control, or DDI1/2 with and without knockdown of RTF2. Error bars represent SEM n=3. See also Figure S2

Article Snippet: Rabbit RTF2 , Proteintech , Cat# 16633-1-AP.

Techniques: Multiplex sample analysis, Control, Biomarker Discovery, Mass Spectrometry, Western Blot, Labeling, Stable Transfection, Expressing, Immunoprecipitation, Knockdown

Journal: Molecular cell

Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

doi: 10.1016/j.molcel.2017.11.035

Figure Lengend Snippet: (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved caspase-3 staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3

Article Snippet: Rabbit RTF2 , Proteintech , Cat# 16633-1-AP.

Techniques: Control, Labeling, Staining, Knockdown

Journal: Molecular cell

Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

doi: 10.1016/j.molcel.2017.11.035

Figure Lengend Snippet: (A) Representative examples of chromosomes in the three classes of metaphases observed in U2OS cells after replication in the presence of low-dose HU or aphidicolin (Aph): normal, damaged, and highly damaged/uncountable. (B) Quantification of the proportion of each of these types of metaphase following 40 h of 0.2 mM HU or 0.4 μM Aph treatment in control and DDI1/2 depleted cells. (C) Quantification of the number of breaks per countable metaphase in U2OS control or DDI1/2 depleted cells. Cells were scored after no treatment, 40 h of 0.4 μM aphidicolin treatment or 40 h of 0.2 mM HU treatment. (D) Quantification of the number of breaks per countable metaphase in control or DDI1/2 depleted cells with or without knockdown of RTF2. Cells were scored after 40 h of 0.4 μM aphidicolin treatment. See also Figure S4

Article Snippet: Rabbit RTF2 , Proteintech , Cat# 16633-1-AP.

Techniques: Control, Knockdown

Journal: Molecular cell

Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

doi: 10.1016/j.molcel.2017.11.035

Figure Lengend Snippet: (A) Immunoblot analysis of whole cell extract to assess phosphorylated RPA32 levels. Control or DDI1/2 depleted cells with or without knockdown of RTF2 were treated with 0.5 mM HU for 20 h, released, and allowed to recover for 8 hours. (B) Quantification of mean nuclear intensity of chromatin-bound RPA staining in cells treated with 20h 0.5 mM HU and extracted with 0.25% Triton-X. (C) Analysis of nascent strand resection in control, DDI1/2-depleted, and BRCA2-depleted cells during 5 h of 4 mM HU treatment with or without the Mre11 inhibitor mirin. Cells were sequentially labeled for 20 minutes each with IdU and CldU. If no resection took place, the CldU/IdU ratio should be 1. Western blot shows levels of BRCA2 after siRNA-mediated depletion. (D) Analysis of DNA damage by alkaline comet assay. Control or DDI1/2 depleted cells were treated with 0.5 mM HU for 20 h, released, and allowed to recover for 8 hours. (E) Analysis of DNA damage by neutral comet assay. Control or DDI1/2 depleted cells were treated with 0.5 mM HU, 10 μM of the ATR inhibitor VE-821, or a combination of the two drugs for 20 h. (F) Western blot showing complementation by HA:RTF2 of the decrease in phospho-RPA signal following 20 h 1 mM HU in RTF2 depleted cells.(G) Graph showing relative endogenous RTF2 mRNA levels in cells treated with RTF2 siRNAs +/− complementation with HA-FLAG:RTF2. qPCR primer F recognizes the 5’ untranslated region of the endogenous transcript only. (H) Quantification of phosphorylated-RPA levels relative to total RPA. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S5 and S6.

Article Snippet: Rabbit RTF2 , Proteintech , Cat# 16633-1-AP.

Techniques: Western Blot, Control, Knockdown, Staining, Labeling, Alkaline Single Cell Gel Electrophoresis, Neutral Comet Assay

Journal: Molecular cell

Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

doi: 10.1016/j.molcel.2017.11.035

Figure Lengend Snippet: Human RTF2 travels with the replisome. We show that when RTF2 is depleted, there is less RPA phosphorylation under replication stress conditions, consistent with a decrease of uncoupling of the replicative helicase and polymerase. Under physiological conditions, RTF2’s function is limited by its DDI1/2-dependent turnover. Both mouse and human RTF2 have been shown to be ubiquitinated and SUMOylated in vivo in high throughput assays. RTF2 also has a putative SUMO interacting domain. It remains to be determined what posttranslational modifications of RTDC1 are necessary for its degradation. Eventually, the turnover signal is turned off and normal replication resumes. In the absence of functional DDI1 and DDI2, RTF2 is not removed from the stressed fork and excess ssDNA accumulates, leading to functional deactivation of that fork and subsequent genomic instability when the replication cannot be completed.

Article Snippet: Rabbit RTF2 , Proteintech , Cat# 16633-1-AP.

Techniques: Phospho-proteomics, In Vivo, High Throughput Screening Assay, Functional Assay

Journal: Molecular cell

Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

doi: 10.1016/j.molcel.2017.11.035

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit RTF2 , Proteintech , Cat# 16633-1-AP.

Techniques: Virus, Recombinant, Multiplex sample analysis, Single Cell Gel Electrophoresis, Protein Quantitation, Software