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. Author manuscript; available in PMC: 2019 Jun 10.
Published in final edited form as: Nat Chem. 2018 Dec 10;11(3):254–263. doi: 10.1038/s41557-018-0187-4

Figure 5 |. Pulldown of hENT1 from red cell membrane fractions using a biotin-rapadocin conjugate or GST-FKBP12-rapadocin complex, and the FKBP-dependence of hENT1 inhibition by rapadocin in cells.

Figure 5 |

a, Affinity pulldown of detergent-solubilized hENT1 by a biotin-rapadocin conjugate and competition by free rapadocin, FK506 or rapamycin. hENT1 was detected by Western blotting. b, Affinity pulldown of hENT1 by GST-FKBP12 in the presence of rapadocin and competition by free rapadocin, FK506 and rapamycin. c, FK506 and rapamycin antagonize rapadocin inhibition of hENT1-mediated thymidine uptake. The dose-response curves were determined using [3H]-thymidine uptake in PK15-ENT1 cells. d, Cyclosporine A and AZD8055 did not antagonize rapadocin inhibition of hENT1-mediated thymidine uptake. The dose-response curves were determined using [3H]-thymidine uptake in PK15-ENT1 cells. e, Knockout of FKBP12 confers resistance to rapadocin. Dose response curves for inhibition of thymidine uptake by rapadocin in wild type and FKBP12 null Jurkat T cells. Representative image of n = 3 independent experiments with similar results. In all graphs, error bars represent s.d.; data are mean ± s.d; n = 3 independent experiments.