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. 2015 Nov 23;112(49):E6752–E6761. doi: 10.1073/pnas.1520957112

Fig. 1.

Fig. 1.

Ii-FRB is not an efficient ER trap for FKBP-tagged proteins due to its interaction with endogenous FKBP13 in the presence of rapamycin (Rap). (A) HeLa cells transiently expressing Ii-FRB-Cer were treated with and without 500 nM rapamycin for 4 h as indicated and subjected to anti-GFP pull-downs. The total lysates and pull-down fractions were analyzed by Western blot for Ii-FRB-Cer and endogenous FKBP13 using anti-GFP and anti-FKBP13 antibodies, respectively. (B) Confocal images of HeLa cells expressing either FKBP12-EGFP-GPI and Ii-FRB-mRFP (rows 1 and 2) or FRB-EGFP-GPI and Ii-FKBP12-mRFP (rows 3 and 4) following incubation with or without rapamycin for 4 h. (Scale bar: 10 μm.) (C) Percentage of cells expressing the indicated ER trap and FRB/FKBP12-tagged GPI-anchored cargo proteins with minimal, partial, and major amounts of EGFP-GPI fluorescence in the ER following 4 h of incubation with rapamycin. At least 50 cells were analyzed for each condition.