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. 2023 Dec 23;300(2):105594. doi: 10.1016/j.jbc.2023.105594

Figure 1.

Figure 1

NanoBRET dilution assay reveals two putative heterodimers, ABCB5β/B6, and ABCB5β/B9. HEK-293T cells were transfected with decreasing amounts of NanoLuc and HaloTag constructs. Transfection started at 1 μg and gradually decreased to 0.125 μg per construct. DMSO refers to the technical negative control to which DMSO is added instead of ligand. Protein pairs serving as negative controls (ABCB2-ABCD1, ABCB3-ABCD1, ABCD1-ABCB5β) (A) and a positive control pair (ABCB2-ABCB3) (B) were tested alongside ABCB5β-ABCB6, ABCB5β-ABCB8, and ABCB5β-ABCB9 (C). Data are presented as mean ± SD. Simple linear regression was used to test whether the amount transfected significantly influenced the NanoBRET ratio (n = 3). p-values are represented on graphs as ∗∗∗p < 0.001, ∗∗p < 0.01, and p > 0.05. For ABCB2-ABCD1 (R2 = 0.8719, F(1,10) = 68.07), ABCB3-ABCD1 (R2 = 0.6144, F(1,10) = 15.94), ABCB5β-ABCD1 (R2 = 0.6661, F(1,10) = 19.95), and ABCB5β-ABCB8 (R2 = 0.7526, F(1,10) = 30.41), the relation between both parameters was statistically significant. For ABCB2-ABCB3 (R2 = 0.0269, F(1,10) = 0.2767), ABCB5β-ABCB6 (R2 = 0.0011, F(1,10) = 0.0113), and ABCB5β-ABCB9 (R2 = 0.0204, F(1,10) = 0.2080), changes in the amount of transfected DNA did not influence the NanoBRET ratio. DMSO, dimethyl sulfoxyde; NanoBRET, nanoluciferase-based bioluminescence resonance energy transfer.