Fig. 7.

Wild-type LSH, but not the ATP-binding-deficient LSH K237Q, can rescue the kinetics of γH2AX in irradiated Lsh^−/− MEFs. (A) Western blots with antibodies against LSH and FLAG show the levels of LSH protein in wild-type (WT) MEFs, Lsh^−/− MEFs infected with empty MSCV lentivirus and cells infected with lentivirus expressing either wild-type or mutant 3× FLAG-tagged LSH protein. MRE11 serves as a loading control. (B) % survival after IR of Lsh^−/− MEFs carrying empty MSCV vector, wild-type LSH and mutant LSH K237Q. The error bars indicate S.D. (C) Lsh^−/− MEFs expressing wild-type LSH, but not cells expressing the K237Q mutant form of LSH, display normal kinetics of γH2AX after IR when compared to control cells. H2AX serves as a loading control. All samples were run on the same gel and detected simultaneously. The numbers on the left indicate molecular weight markers in kDa. (D) Immunostaining with anti-FLAG and anti-53BP1 antibodies detect normal localisation of 53BP1 to DNA damage foci in Lsh^−/− MEFs expressing wild-type LSH. Irradiated Lsh^−/− MEFs infected either with empty MSCV virus or expressing LSH K237Q show diffuse staining for 53BP1. The scale bars represent 10 µm.