Fig. 2.

Targeting the ERα proximal proteome using epitope tag antibody.A, schematic of BAR workflow targeting ERα-Flag. Cells stably expressing ERα-Flag under the control of a Tet-Off promoter (MCF7-TO cells) were labeled with biotin using epitope tag antibody to vehicle the proximity reaction. Cells were treated with doxycycline (DOX) to silence ERα-Flag expression in control samples. B, immunofluorescence images of ERα-Flag BAR. MCF7-TO cells treated with or without DOX were biotinylated as in (A). Biotin colocalized with ERα-Flag and biotinylation resulted in nuclear localization. Little biotinylation was observed in the negative control. Nuclei were detected by DAPI (gray); ERα-Flag was visualized by Alexa Fluor 488 dye (gold). Biotinylated proteins were detected by streptavidin-conjugated Alexa Fluor 647 dye (magenta). C, western blot analysis of ERα-Flag BAR biotinylated proteins. Labeled cells were lysed and incubated with streptavidin beads. ERα-Flag was efficiently immunoprecipitated (SA-IP) among biotinylated proteins (Input). D, multi scatter plot of proteins identified in the ERα-Flag BAR experiment. The log₂-transformed label-free quantification intensities are plotted against each other. Pearson correlation coefficients for each comparison are reported in the upper left corner of the plots. E, volcano plot showing proteins significantly enriched in biotinylated versus control samples. Statistically significant proteins (student t test q-value <0.01) are shown in gold; proteins nonstatistically significant are shown in gray. ERα well-known interacting proteins are highlighted. F, heatmap showing ERα known interactors enriched versus negative control. Proteins are colored according to their abundance.