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. 2017 Oct 31;11:337. doi: 10.3389/fncel.2017.00337

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Copyright © 2017 Pratt, Kuljis, Homanics, He, Kolodieznyi, Dudem, Hollywood, Barth and Bruchez.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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FAP-tagged BKα subunits coassemble with untagged subunits. (A) Crude membrane fractions from brain, kidney, and bladder were probed for BKα and HA tag. Untagged BKα migrated at 125 kDa and FAP-BKα migrated at 155 kDa. Both bands were present in brain, with HA reactivity in the 155 kDa band. To visualize weak signal in bladder smooth muscle, a long exposure is shown; however, HA reactivity was undetectable in these preparations. Quantification of total BKα normalized to NaK-ATPase is shown below. n = 2 biological replicates, error bars are SD. ^∗∗P ≤ 0.01 compared to WT, one-way ANOVA with Tukey’s multiple comparison test. (B) Schematic for five possible configurations of BKα:FAP-BKα tetramerization ranging from homomeric FAP-BKα to channels lacking FAP; expected densitometry ratios are given for each condition. (C) Immunoprecipitation using antibodies directed against BKα C-terminus yielded FAP-tagged and untagged channels. FAP-tagged channels showed HA reactivity, which was depleted in flowthrough. (D) Immunoprecipitation using antibodies against HA yielded tagged and untagged channels. Only the high M.W. bands were HA reactive in input and IP. (E,F) Ratios of high and low (155 kDa:125 kDa) BKα-reactive band densitometry in whole lysates (E, n = 4 animals, 6 protein preparations) and HA immunoprecipitated samples (F, n = 2 animals, 3 protein preparations). Significance tested using unpaired t-test with Welch’s correction.