β2 protein co-assembles with Slo1/BK protein, but in the Slo1 KO, β2 protein, but not β2 message, is diminished. (A) Total proteins were initially immunoprecipitated with NeuroMab anti-Slo1 Ab or the NeuroMab N53/32 BKβ2 Ab (nAB) and then visualized either with NeuroMab N33 Slo1 Ab (top) or with NeuroMab BKβ2 N53/32. Slo1 protein is unaffected by KO of the β2 protein in all tested tissues (top), whereas β2 (red arrow) is markedly reduced after KO of Slo1 (bottom). nAb-IP, which corresponds to omission of Ab during IP but inclusion of IP beads, is ineffective at IP of either Slo1 or β2. The bottom panels establish that, in WT tissues, IP of Slo1 also pulls down β2 protein. (B) Total proteins from the indicated tissues and genotypes were initially immunoprecipitated with Alomone anti-BKbeta2 Ab, treated with N-glycanase, and then visualized with NeuroMab anti-BKbeta2 N53/32. β2 protein (red arrow) is markedly reduced in tissues from slo1^−/− mice and totally absent in tissues from kcnmb2^−/− mice. (A and B) Molecular mass is indicated in kilodaltons. WB, Western blot. (C) Relative mRNA abundance in mouse adrenal (whole adrenals) from one WT and one Slo1 KO mouse was determined in triplicate for β1, β2, β3, β4, and Slo1, with normalization to β-actin message. (D) Relative abundance of different subunits for WT and slo1^−/− mice is shown for cerebellum.