Relative amounts ofα7 and non-α7 mt-nAChR subtypes in BEP2D cells before and after malignant transformation. The transformation was achieved due to 48 h exposure to 2 μg/ml NNK followed by 5 passages, and confirmed in the in vitro and in vivo tumorigenicity assays (see Methods). In radioligand-binding assay, the numbers of α7 and non-α7 nAChRs were estimated by the amounts of specific binding of [3H] αBtx and [3H] epibatidine, respectively. Quadruplicate samples of mitochondria from control (intact) and NNK-transformed BEP2D cells were incubated for 30 min at 0o C with saturating concentrations of the radioligands. The specific binding was calculated by subtracting the non-specific binding from total binding. In sELISA, the mitochondrial proteins were probed with anti-α7 or anti-α3 antibodies, as detailed in Methods. Results of both assays are expressed as fold of control, taken as 1. Data are mean + SD. Asterisk = p < 0.05 compared to respective controls.