Skip to main content
. 2022 Mar 24;298(5):101865. doi: 10.1016/j.jbc.2022.101865

Figure 3.

Figure 3

Progressive acceptor photobleaching provides insight into regulatory complex stoichiometry.A, images of cells expressing NKA and PLM labeled with mCerulean (mCer) and YFP, respectively, before and after acceptor selective photobleaching. The scale bar represents the size of 10 μm. B, in a control experiment, donor (mCer) fluorescence intensity increased as acceptor (Venus) was progressively photobleached. C, replotting data (as in B) reveals the relationship between donor and acceptor fluorescence intensity. The relationship is linear when one acceptor is present, for example, mCer separated from Venus by five amino acids (C5V, black) but is curved when there are multiple acceptors participating in FRET (VCV, red). D, the regulatory complex contains multiple acceptor-labeled PLM in complex with donor-labeled α (n = 56). E, the complex contains multiple acceptor-labeled β (n = 42). F, the α-α complex produced a linear donor/acceptor relationship, suggesting a single acceptor-labeled α in complex with donor-labeled α (n = 150). NKA, Na/K-ATPase; YFP, yellow fluorescent protein.