ps 345 chk1  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

doi: 10.1371/journal.pone.0038009

Figure Lengend Snippet: (A) The signaling pathway that controls entry into mitosis. See text for details. (B) Arrested Geminin-deficient cells exhibit increased phosphorylation of Cdc2 on Y 15 and increased levels of B-type cyclins; and these changes are reversed by over-expressing either Cdc2 AF or Cdc25 S287A . Two-cell embryos were left uninjected (CO), injected on both sides with anti-Gem MOs (a-Gem MOs), or injected with anti-Gem MOs followed by RNA encoding Cdc2 AF or Cdc25 S287A . At stage 10.5, phosphorylated Cdc2 and cyclin B1 levels were determined by immunoblotting. Load, cross-reacting band serving as a loading control. (C) The cell cycle arrest is reversed by over-expressing Cdc25 WT , Cdc25 S287A , Cdc2 AF , or Chk1 DA . One cell of a two-cell embryo was injected with anti-Geminin MOs and RNA encoding the indicated proteins. The plot shows the percentage of embryos in which cell division was restored in the injected area at stage 10.5. (D–M) One cell of a two-cell embryo was injected with anti-Gem MOs and/or RNA encoding the indicated proteins. LacZ RNA was co-injected as a lineage tracer. At stage 10.5 Xbra RNA was visualized by in situ hybridization (purple) and beta galactosidase activity was visualized by staining with Xgal (blue). (N) Both sides of a two-cell embryo were injected with anti-Geminin MOs and/or RNA encoding the indicated proteins. At stage 10.5, RNA was extracted and the amount of Xbra mRNA was measured by RT-PCR (gray bars). In a parallel experiment, one cell of a 2-cell embryo was injected in the same way along with LacZ as a lineage tracer. At stage 10.5 Xbra was visualized by in situ hybridization and the percentage of embryos showing normal Xbra expression was determined (black bars).

Article Snippet: Phosphospecific antibodies against pS 139 H2A.X and pS 345 Chk1 were purchased from Cell Signaling.

Techniques: Expressing, Injection, Western Blot, In Situ Hybridization, Activity Assay, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

doi: 10.1371/journal.pone.0038009

Figure Lengend Snippet: Two-cell Xenopus embryos were injected on both sides with anti-Geminin MOs and/or RNA encoding the indicated proteins. The levels of S 345 -phosphorylated Chk1, S 139 -phosphoryated H2A.X, and Geminin were determined by immunoblotting.

Article Snippet: Phosphospecific antibodies against pS 139 H2A.X and pS 345 Chk1 were purchased from Cell Signaling.

Techniques: Injection, Western Blot

Journal: Nucleic Acids Research

Article Title: ATR-dependent pathways control hEXO1 stability in response to stalled forks

doi: 10.1093/nar/gkm1052

Figure Lengend Snippet: ATR-dependent control of hEXO1 stability in response to stalled replication. ( A ) HEK-293, U2OS, HeLa and MEF cells were grown in the presence or the absence of 2 mM HU for 24 h. Whole cell extracts were immunoprecipitated with the rabbit polyclonal antibody F-15 and resolved on a 8% SDS–PAGE. Human and mouse EXO1 were visualized with the monoclonal antibody Ab-4. MSH6 and IgG(H) were used as controls for the input and the quality of the immunoprecipitation, respectively. ( B ) Upper panels: immunoprecipitation of hEXO1 from HEK-293T cells left untreated (lane 1) or treated for 8 h with either 10 μM KU-55933 (lane 2), 4 mM caffeine (lane 3), 2 mM HU (lane 4), 4 mM caffeine followed by 2 mM HU (lane 5) or 10 μM KU-55933 followed by 2 mM HU (lane 6). hEXO1 was detected as described above. The signal given by IgG(H) was used as control for the quality of the immunoprecipitation. Lower panels: western blot analysis of phosphorylated CHK1 in whole cell extracts was used as read-out for the cellular response to HU. TFIIH was used as loading control. ( C ) HEK-293T cells were stably transfected with EV, ATR or ATM shRNA constructs. hEXO1 was immunoprecipitated from cells left untreated or treated with HU for 16 h. The signal given by IgG(H) was used as control for the quality of the immunoprecipitation. ATR and ATM expression was monitored to assess the extent of downregulation by shRNA. TFIIH served as loading control. The data shown represent one of three independent experiments. ( D ) ATR-Seckel cells at early or late passages were either left untreated or treated with HU for 16 h. hEXO1 was immunoprecipitated as described above. TFIIH and IgG(H) were used as controls for the input and the quality of the immunoprecipitation, respectively. The expression of ATR was examined by western blot analysis. ( E ) AT or AT + ATM fibroblasts were treated in the presence or the absence of HU and the expression of hEXO1 was assessed following immunoprecipitation as described above. TFIIH and IgG(H) were used as controls for input and quality of the immunoprecipitation, respectively.

Article Snippet: Antibodies to CHK1-pS 345 , CHK2-pT 68 and p53-pS 15 were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Stable Transfection, Transfection, shRNA, Construct, Expressing