kap 1 ser p 15 rabbit polyclonal  (Novus Biologicals)

Journal: The Journal of Biological Chemistry

Article Title: ATM Protein-dependent Phosphorylation of Rad50 Protein Regulates DNA Repair and Cell Cycle Control *

doi: 10.1074/jbc.M111.258152

Figure Lengend Snippet: The mutant form of Rad50 (S635G) is defective in complementing DNA damage-induced SMCI phosphorylation. A, shown is the effect of wild type and mutant Rad50 (S635G) on radiation-induced phosphorylation of ATM-dependent substrates. Rad50-deficient fibroblasts were transfected with Rad50 constructs as in Fig. 1E. Extracts were prepared from non-irradiated and irradiated (5 Gy) cells at 15 min post-irradiation. Proteins were separated on SDS-PAGE, and membranes were immunoblotted with antibodies against ATM-downstream substrates and also phosphorylated forms of these substrates. Extracts from irradiated and unirradiated cells were blotted with various ATM substrate antibodies as indicated. B, shown is the effect of Rad50 constructs on radiation-induced phosphorylation of ATM-dependent substrates after exposure to 1-Gy radiation and incubation for 15 min. Experimental conditions and analysis were the same as used in A above but with lower dose radiation. C, investigation of the expression of the germ line mutant Rad50 (X1313Y extX*66) form. Rad50-deficient cells were transfected with vector, Rad50WT, and Rad50 mutant (S635G). Stable cell lines were established as described above, and extracts were prepared before separation on 4.2% SDS-PAGE and immunoblotting with Rad50 antibodies. D, interaction of Rad50 with SMC1 is shown. Extracts were prepared from control fibroblasts before adding ethidium bromide (100 ng/ml) followed by immunoprecipitation (IP) with Rad50 antibody and immunoblotting with Rad50 and SMC1 antibodies. Extracts were also immunoprecipitated with nonspecific sera as a control. E, shown is use of Rad50-GST constructs spanning the full length of the protein to determine the region interacting with SMC1. Control fibroblast cells were treated with 5-Gy radiation and harvested 30 min later. Total protein extracts (1 mg) were either incubated with GST alone or with 5 μg of each Rad50 GST fusion protein. Protein complexes were fractionated by 4.2% SDS-PAGE and then analyzed by immunoblotting with specific antibodies against Rad50 and SMC1. Gels of the GST fusion proteins were stained with Coomassie stain (lower panel). Under these conditions GST alone had migrated off the gel. F, Rad50 regulates ATM-dependent SMC1 phosphorylation in response to DNA damage. A GST fusion protein corresponding to the kinase domain of ATM (GST12) was employed in pulldown experiments to look for interaction with Rad50, SMC1, and the phosphorylated form of SMC1 (SMC1 Ser(P)-957 (pS957)). Proteins were determined by immunoblotting on control extracts bound by ATM GST12 and separated on SDS-PAGE. GST-only beads were used as a negative control, and under these conditions GST had migrated from the gel. G, mutant Rad50 abrogates ATM-dependent phosphorylation of SMC1 without interfering with interaction between SMC1 and Rad50. Rad50-transfected cells were employed as described in A. Rad50 immunoprecipitates were resolved on 6% SDS-PAGE followed by immunoblotting with anti-Rad50 Ser(P)-635 (pS635), SMC1 Ser(P)-957, SMC1, and Rad50 antibodies. H, wild-type Rad50 is required for DNA damage-induced interaction of SMC1 Ser(P)-957 with chromatin. Chromatin was fractionated from Rad50-deficient-transfected cells as described under “Experimental Procedures.” These fractions were resolved in 6% SDS-PAGE and immunoblotted with anti-SMC1 Ser(P)-957, SMC1, and Rad50 antibodies.

Article Snippet: Antibodies to SMC1, NBS1, KAP-1, and KAP-1 Ser(P)-15 (rabbit polyclonal) were purchased from Novus Biologicals; anti-FLAG and anti-β actin were from Sigma; anti-rabbit p53 and anti-mouse p53 Ser 15 were from Cell Signaling Technology; anti-mouse monoclonal antibodies to Rad50, SMC1 Ser-957, and NBS, Ser 343 were from Upstate Biotechnology; anti-rabbit Chk2 and Chk2 pT68 were from Abcam; anti-rabbit ATM Ser-1981 was from Rockland, and anti-ATM and Mre11 (mouse monoclonal antibodies) were from GeneTex.

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Construct, Irradiation, SDS Page, Incubation, Expressing, Plasmid Preparation, Stable Transfection, Western Blot, Control, Immunoprecipitation, Staining, Negative Control