Trip br3

MCF7 and MCF10A cells were grown to ~60% confluence on sterilized round type cover glasses in DMEM or DMEM/F12 with or without serum for 48 h. Cells were fixed with 4% formaldehyde in PBS for 15 minutes, washed with PBS, and incubated with TRIP-Br3 (Abcam, Cat. ab107944) primary antibody overnight at 4°C. Cells were then washed with PBS and incubated with Rabbit IgG (H+L) secondary antibody (Jackson Immuno Research Laboratories, Cat. 111–025-003, U.S.A) conjugated to Alex Fluor dyes for 1 h at room temperature in the dark. Cells were washed with PBS and the coverslips were mounted on glass slides with VECTASHIELD anti-fade mounting medium containing 4′6-diamidino-2-phenylindole (DAPI; Vector Laboratories, U.S.A). Samples were imaged using a laser scanning confocal microscope (Leica, Germany) and fluorescence was analyzed.

Immunoblotting analysis was performed as previously described [29 (link)]. Antibodies used in this study were TRIP-Br1 (Enzo Life Sciences, Cat. ALX-804–645), TRIP-Br2 (Abcam, Cat. ab87150), TRIP-Br3 (Abcam, Cat. ab107944), SERTAD3 (Abcam, Cat. ab107728), XIAP (Cell Signaling Technology, Cat. #2042), Bax (SantaCruz Biotechnology, Cat. sc-20067), LC3 (Enzo Life Sciences, Cat. ALX-803–082), LaminB (SantaCruz Biotechnology, Cat. sc-6216), Anti-Flag (Sigma-Aldrich, Cat. F2555), and γ-tubulin (SantaCruz Biotechnology, Cat. sc-7396).