(A) Map of the ARMC2 gene. The open arrowhead indicates the position of the insertion in the CLiP mutant LMJ.RY0402.155726. (B) Average swimming velocity of the strains as indicated. The standard deviation and the number of cells analyzed are indicated. (C) Western blot analysis of isolated flagella of control, the RSP3 mutant pf14, armc2, and the armc2 ARMC2-mS rescue strain with antibodies to RSP3 and as loading controls, the outer arm dynein subunit IC2 and IFT139. Note accumulation of IFT139 in pf14 and armc2 as previously reported for paralyzed central pair mutants of Chlamydomonas (Lechtreck et al., 2013; see Figure 2—figure supplement 1B). (D) Western blot analysis of isolated flagella of control, pf27, and the pf27 ARMC2-3xTAG rescue strain with antibodies to RSP3, anti-HA, and anti-IC2, as a loading control. Anti-HA was used to document expression of ARMC2-3xTAG (the 3xTAG encompasses a triple HA tag). A representative Western blot of three biological replicates is shown. (E) DIC and TIRF imaging of live cells showing the distribution of RSP3-NG in the armc2 pf14 RSP3-NG mutant (a–f) and the armc2 pf14 ARMC2-mS RSP3-NG rescue strain (g–i). Bars = 10 µm. (F) Immunofluorescence staining of methanol-fixed control (a–c), pf27 (d–f), and pf27 ARMC2-3xTAG (g–i) cells stained with anti-acetylated-α-tubulin (a, d, g) to visualize flagella and affinity-purified anti-RSP3 (b, e, h); merged images are shown in the bottom row (c, f, i). Arrowheads in e, residual RSP3 near the proximal end of the pf27 flagella. The bright signal of the cell body likely results from unspecific binding of the anti-RSP3 antibody and chlorophyll autofluorescence. Bar = 10 µm.