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Fig. 4 CrCYB5D1 is a radial spoke protein. (A) Localization of mC::CrCYB5D1 and IFT46::YFP in the flagella of Crcyb5d1 mC::CrCYB5D1 using 3D-SIM. (Scale bars, 2 μm.) (B) Immunoblot analysis of equal amounts of protein from whole-cells (WC), cell bodies (CB), and isolated flagella (Fla) of Crcyb5d1 mC::CrCYB5D1 probed with antibodies against RSP1, mCherry, and α-tubulin. (C) Immunoblot analysis of flagella, axonemes, and membrane plus matrix isolated from Crcyb5d1 mC::CrCYB5D1 probed with antibodies against FMG-1, RSP1, mCherry, and α-tubulin. FMG-1 is a flagellar membrane marker and RSP1 acts as a radial spoke marker. (D) Immunoblot analysis of a NaCl extract and subsequent KI extract of axonemes from Crcyb5d1 mC::CrCYB5D1 probed with antibodies against PF20, IC2, RSP1, and mCherry. PF20 was used as a central pair marker and IC2 as an outer arm dynein marker. Immunoblot analysis of equal amounts of protein from whole-cells, cell bodies, and isolated flagella of pf20 mC::CrCYB5D1 (E) and pf28pf30 mC::CrCYB5D1 (F) probed with antibodies against mCherry and α-tubulin.