Fig. 1

LCK interacts with RAD51/BRCA1 in response to DNA damage and stabilizes RAD51/BRCA1 (A) Structure of MYC labelled LCK construct. LCK Y394F, LCK K273R, LCK Y192F mutants were generated by site directed mutagenesis. Mutants were transduced in CP70 cells on LCK CRISPR KO background. B Myc tagged LCK expressing CP70 cells were treated with vehicle (DMSO) or etoposide for 24 h, washed to remove etoposide and incubated for an additional 24 h, then harvested, lysed, and nuclei were purified. Extracts were Immunoprecipitated with Myc followed by immunoblotting for Myc, BRCA1, and RAD51. C Myc tagged LCK expressing SKOV3 cells were likewise treated with vehicle or etoposide. Myc protein was immunoprecipitated and immunoblotted for LCK, RAD51, and BRCA1. D LCK overexpressing SKOV3 cells were treated with etoposide. RAD51 was immunoprecipitated from total extracts followed by immunoblotting for LCK and RAD51. E CP70 cells (MYC tagged LCK, LCK Y394F, LCK K273R, and LCK Y192F) were treated with etoposide/DMSO for 24 h, washed free of Etoposide and incubated for an additional 24 h. Cells were harvested and nuclei isolated. Extracts were immunoprecipitated with MYC antibodies followed by immunoblotting for LCK, RAD51, and BRCA1 proteins. F Schematic of LCK binding partners in response to DNA damage. G CP70 EV and CP70 LCK OE cells were treated with cycloheximide over a 6 h period and extracts collected at 0, 1, 2, 3, 5, and 6 h. Cells were harvested followed by immunoblotting for RAD51 and BRCA1 protein expression. Each experiments were repeated for at least three times