GST and GST-RAP fusion protein were expressed in bacteria and then purified. Briefly, recombined plasmid was expressed in E. coli Transetta (DE3) cells. At OD600 = 0.5, protein expression was induced with 1 mM IPTG (Solarbio) for 5 h at 25°C. Bacteria were lysed and sonicated in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.5% Triton X-100, PMSF, DTT, lysozyme, and protease inhibitor cocktail (Sigma-Aldrich) at 4°C. After centrifugation at 12,000 g for 15 min at 4°C, the supernatant was used for the protein purification with GST resin (TransGen Biotech) according to the manufacturer’s instructions. Elution of recombinant protein was performed under mild, non-denaturing condition using reduced glutathione. Then, the fractions were collected and analyzed with SDS-PAGE followed by Coomassie brilliant blue staining.
Breast cancer cells were treated with 200 nM GST-RAP fusion protein for 72 h followed by immunofluorescent staining. Briefly, cells grown on slides were fixed with 4% paraformaldehyde. After blocking, slides were stained with anti-GST antibody (Proteintech, 10000-0-AP). Slides were then incubated with Alexa-488 Goat anti-Rabbit IgG (Invitrogen, A11008). Nuclei were stained with DAPI (Thermo Fisher Scientific).
Breast cancer cells were treated with 200 nM GST-RAP fusion protein for 72 h followed by immunofluorescent staining. Briefly, cells grown on slides were fixed with 4% paraformaldehyde. After blocking, slides were stained with anti-GST antibody (Proteintech, 10000-0-AP). Slides were then incubated with Alexa-488 Goat anti-Rabbit IgG (Invitrogen, A11008). Nuclei were stained with DAPI (Thermo Fisher Scientific).