Fig. 4. (A) Schematic representation of the membrane reporter translocation assay. (B) Cells treated with either rapamycin or BORap and irradiated at 0 hours followed by YFP imaging at 2 hours. Scale bar equals 10 μM. (C) Quantification of the YFP signal intensity before and after irradiation (90 s), showed reduced fluorescence in BORap-treated cells, while all other conditions show little to no decrease in fluorescent signal. Average fluorescence intensity in 20 cells is shown normalized to pre-irradiated controls, with error bars representing standard deviations. Statistical analysis was performed in Prism using a two-way ANOVA test to compare irradiated samples for each treatment condition to pre-irradiated samples. P values less than or equal to 0.0001 are represented by ****, while all other comparisons are not significant. (D) HEK293T cells expressing the CFP and YFP constructs were treated with 20 μM of rapamycin or BORap prior to irradiating with 530 nm light. Western blot analysis using anti-GFP, anti-GAPDH, and anti-nucleolin antibodies showed a light-induced band decrease with BORap but not rapamycin treatment. (E) Triplicate experiments were performed and quantified using FIJI. Band intensity of FKBP, FRB, and nucleolin for each irradiation timepoint were normalized to the rapamycin-treated, non-irradiated sample and averaged. Statistical analysis was performed as above, comparing irradiated to non-irradiated samples within each treatment condition (10, 30, or 60 s vs. 0 s). P values less than or equal to 0.001 are represented by ***, less than or equal to 0.01 by **, while comparisons of rapamycin-treated samples (for FKBP, FRB, and nucleolin) and comparison of BORap-treated samples (for nucleolin) are not significant.