Journal: Nucleic Acids Research

Article Title: SUMOylation mediates CtIP’s functions in DNA end resection and replication fork protection

doi: 10.1093/nar/gkaa1232

Figure Lengend Snippet: CtIP is a target for SUMO-2 modification. ( A ) In vitro reactions assembled using a SUMO-2 conjugation kit and recombinant glutathione S-transferase (GST)-tagged human CtIP were resolved by SDS-PAGE and immunoblotted. Despite even input of UBC9 into the reactions, less UBC9 was consistently detected by immunoblot in Reaction C; we speculate the antibody used preferentially recognizes auto-SUMOylated UBC9, which is absent when ATP is eliminated from the reaction (Reaction C). The solid line defines where an intervening lane was spliced out of the image. The immunoblot shown is representative of six independent experiments. ( B ) HeLa cells expressing 10XHis-tagged SUMO-2 (His 10 -SUMO-2) or parental HeLa cells were portioned into input and His pull-down (His PD) fractions and processed as whole cell extracts or Ni-NTA affinity purifications, respectively, then resolved by SDS-PAGE. Shown is a representative result of two independent experiments. * indicates a non-specific immunoreactive band. Corresponds to . ( C ) As in (B) but with His 10 -SUMO-2 cells only, representative of at least three independent experiments. ( D ) 10% of a cell pellet of HeLa His 10 -SUMO-2 cells was lysed as the whole cell extract (top panel); the remainder was fractionated for chromatin enrichment. 1/9 of each of the resulting soluble (sol) and chromatin-enriched pellet (pel) fractions was saved to represent the input prior to His PD. The remainder was processed for His PD, and all fractions were resolved by SDS-PAGE. For the first run (middle panel), the His PD fractions contain 32X more of the starting amount of sample relative to the input. In the second run (bottom panel), the pellet fraction (for input and His PD portions) was loaded to represent a similar starting amount of CtIP as the soluble fraction. Shown is a representative result of two independent experiments.

Article Snippet: Unless indicated otherwise, cell lysates were prepared by resuspending cell pellets into 2× SDS-PAGE Sample Buffer (125 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.1% bromophenol blue, 5% 2-mercaptoethanol), heating for 2 min at 95°C, and sonicating with a Model 705 Sonic Dismembrator fitted with a microtip probe (Fisher Scientific), generating whole cell extract.

Techniques: Modification, In Vitro, Conjugation Assay, Recombinant, SDS Page, Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: SUMOylation mediates CtIP’s functions in DNA end resection and replication fork protection

doi: 10.1093/nar/gkaa1232

Figure Lengend Snippet: Analysis of CtIP SUMOylation status in S phase and in response to double-strand breaks and replication stress. (A, B, D, E, F) HeLa His 10 -SUMO-2 cells were treated as indicated, portioned into input and His PD fractions and processed accordingly before SDS-PAGE and immunoblotting. Chk2 and Chk1 phosphorylation were used as readouts for the induction of DSBs or replication stress, respectively. ( A ) Asynchronous cells were subjected to 10 Gy of IR or not and allowed to recover for 1 h. Shown is a representative result of three independent experiments. * indicates a non-specific immunoreactive band. Corresponds to . ( B ) Asynchronous cells were exposed to 10 Gy of IR and allowed to recover for 1 h, or subject to CPT, etoposide (etopo), or phleomycin (phleo) at the indicated concentrations for 1 h. Shown is a representative result of four independent experiments. CtIP typically exhibits smearing upon treatment with high dose CPT and etoposide (input fraction). ( C ) HeLa His 10 -SUMO-2 cells were left asynchronous (async) or synchronized by double thymidine block and released for various timepoints to reach different cell cycle phases, then a portion was processed for DNA content analysis. Shown are the means from three independent experiments. ( D ) As in (C), but this time processed for immunoblot of input and His PD fractions. Shown is a representative result of three independent experiments. ( E ) Asynchronous cells were subject to 25 μM phleomycin for 1 h or the indicated replication stress inducing agents for 4 h. 0.8% H 2 O served as the vehicle control; aphidicolin (aphid); hydroxyurea (HU). Shown is a representative result of three independent experiments. ( F ) Asynchronous cells were treated with 2 mM HU for the indicated timepoints or not treated for 4 h. Shown is a representative result of at least three independent experiments.

Article Snippet: Unless indicated otherwise, cell lysates were prepared by resuspending cell pellets into 2× SDS-PAGE Sample Buffer (125 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.1% bromophenol blue, 5% 2-mercaptoethanol), heating for 2 min at 95°C, and sonicating with a Model 705 Sonic Dismembrator fitted with a microtip probe (Fisher Scientific), generating whole cell extract.

Techniques: SDS Page, Western Blot, Blocking Assay

Journal: Nucleic Acids Research

Article Title: SUMOylation mediates CtIP’s functions in DNA end resection and replication fork protection

doi: 10.1093/nar/gkaa1232

Figure Lengend Snippet: SUMOylation of CtIP in S phase is dependent on cyclin-dependent kinase and ATR activities and an interaction with PCNA. (A–E, H) HeLa His 10 -SUMO-2 cells were treated as indicated, portioned into input and His PD fractions and processed accordingly before SDS-PAGE and immunoblotting. * indicates an exogenous CtIP immunoreactive band that is not of interest; we speculate it is either lower molecular weight SUMO-2-modified CtIP or unmodified tagged-CtIP retained in the His PD fraction due to overexpression. ( A ) HeLa His 10 -SUMO-2 cells were transfected with FLAG-tagged wildtype (WT) CtIP or a C-terminal truncation mutant (D6) (see ) or mock transfected. Shown is a representative result of four independent experiments. ( B ) HeLa His 10 -SUMO-2 cells were synchronized by double thymidine block and released to mid-S phase for 1 h in plain media, then for 2.5 h in the presence of 0.1% DMSO (vehicle control), 25 μM roscovitine, 2.5 μM AZD5438, or 10 μM RO-3306. Asynchronous cells (async) were treated in 0.1% DMSO for 2.5 h. Shown is a representative result of three independent experiments. ( C ) HeLa His 10 -SUMO-2 cells were synchronized to mid-S phase. 24 h prior to harvest, they were transfected with GFP-CtIP-WT or substitution mutants at residue T847, or mock transfected. Shown is a representative result of two independent experiments. ( D ) HeLa His 10 -SUMO-2 cells were left asynchronous or synchronized by double thymidine block and released for 3 h to mid-S phase in the presence of 0.2% DMSO, 10 μM KU-55933 (ATM inhibitor), 20 μM ETP-46464 or 20 μM VE-821 (ATR inhibitors), or 1 μM NU-7441 (DNA-PKcs inhibitor). Shown is a representative result of at least four independent experiments. ( E ) HeLa His 10 -SUMO-2 cells were cells were left asynchronous or synchronized to mid-S phase. 24 h prior to harvest, they were transfected with HA-tagged WT-CtIP or an alanine substitution mutant at residues S664, S679 and S745, or mock transfected. Shown is a representative result of two independent experiments. ( F ) U-2 OS cells were co-transfected with RFP-PCNA and either GFP empty vector or GFP-CtIP and processed for immunoprecipitation (IP) of GFP. Prior to IP, a portion of lysate was saved as an input control. ( G ) As in (F), except cells were depleted of endogenous CtIP by siRNA, then co-transfected with RFP-PCNA and either GFP empty vector, GFP-CtIP-WT or -Δ515–518. Shown is a representative result of at least six (F) or three (G) independent experiments. ( H ) HeLa His 10 -SUMO-2 cells were synchronized to mid-S phase. 24 h prior to harvest, they were transfected with GFP-CtIP-WT or the Δ515–518 deletion mutant. Shown is a representative result of two independent experiments.

Article Snippet: Unless indicated otherwise, cell lysates were prepared by resuspending cell pellets into 2× SDS-PAGE Sample Buffer (125 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.1% bromophenol blue, 5% 2-mercaptoethanol), heating for 2 min at 95°C, and sonicating with a Model 705 Sonic Dismembrator fitted with a microtip probe (Fisher Scientific), generating whole cell extract.

Techniques: SDS Page, Western Blot, Molecular Weight, Modification, Over Expression, Transfection, Mutagenesis, Blocking Assay, Plasmid Preparation, Immunoprecipitation