Figure 1
From: MED1, a novel binding partner of BRCA1, regulates homologous recombination and R-loop processing
MED1/BRCA1 complex formation and evaluation of BRCT domain transcriptional activity. (a) Immunoprecipitation (IP) of U2OS cells. Cells were collected, washed with PBS, and dissolved in RIPA buffer to prepare whole-cell extracts. Antibodies (control IgG, anti-BRCA1, anti-MED1) were added to the supernatant and incubated, and Protein G Sepharose Fast Flow beads were added to the supernatant to purify the immune complex. Proteins bound to the beads were extracted, run on 10% SDS-PAGE, and subjected to western blotting, which confirmed the formation of a complex between MED1 and BRCA1. (b) GST-pulldown assay using U2OS cells. The pGEX 4T-1 or pGEX 4T-1 BRCT vectors were transformed into Escherichia coli and cultured. IPTG was added to the culture medium to induce protein expression, E. coli was harvested, and proteins were extracted. The binding of MED1 to the BRCT region was confirmed by western blotting and protein affinity columns. (c) Luciferase assay using 293T cells. 293T cells were seeded into 12-well dishes and transfected with each plasmid for 24 h. Firefly luciferase activity was measured simultaneously using the Dual Luciferase Reporter System (Promega, Madison, WI, USA). Renilla luciferase activity was also measured for normalization. MED1 enhanced the transcriptional activity of GAL4-BRCT by approximately 2.4-fold. Unpaired t test, *p < 0.05, **p < 0.01.
