Figure 3

BK channel clusters appear at E18 during hair cell maturation. (A) Western blot analysis on membrane-bound protein from chick brain reveals a 120 kDa band, near the predicted weight of the minimal BK α variant (127 kDa; no inclusion of alternative exons). A fainter, lower-weight band is also apparent. No other spurious bands were observed in the blot. Both bands were absent when the primary antibody was preadsorbed with the immunogen. (B) Isolated hair cells from E10 to posthatch basilar papilla are shown labeled with antibodies to BK channels (Alomone). The apical surface and hair bundle are outlined to show orientation of the cell (dotted lines). Punctate clusters appear at E18. Bright label at E10 to E12 is inside the cell at the level of the nucleolus. (C) Several preparations were triple labeled for anti-BK (Alomone; red), hair bundles (phalloidin; green), and a nuclear stain (Hoechst; blue). Two examples for each age are shown (left two columns) along with no primary controls (far right column). Anti-BK clusters appear supranuclear at E18 and subnuclear from E20 onward. Scale bars = 10 μm.