Fig. 1

Characterization of the H6PD-BirA*-HA expressing MDA-MB-231 clone. The selected MDA-MB-231 cell clone expressing H6PD-BirA*-HA was analyzed by immunofluorescence and immunoblotting (A–C). A Confirmation of H6PD-BirA*-HA localization in the endoplasmic reticulum lumen using immunofluorescence in MDA-MB-231 cells stably expressing the H6PD-BirA*-HA fusion protein and in parental control cells. Staining was performed using rat anti-HA antibody, rabbit anti-CANX antibody, and the corresponding goat anti-rat Alexa-555 and goat anti-rabbit Alexa-488 secondary antibodies for visualization. In addition, nuclei were stained using Hoechst-33342. B Immunoblotting of lysates from parental MDA-MB-231 and H6PD-BirA*-HA expressing cells incubated for 72 h with or without 50 µM biotin. Membranes were probed with HRP labeled streptavidin (Strep-HRP) to detect biotinylated proteins. β-Actin served as loading control. C Cells were incubated with 50 µM biotin for 72 h where indicated. Biotinylation was qualitatively assessed using streptavidin Alexa-488 antibody. Representative images are shown, scale bars 15 µm