Anti mneongreen

For immunofluorescence, whole cells were washed in PBS, then spread on poly-lysine–coated slides. For cytoskeleton preparations, cells were first extracted with 0.25% NP40 in PIPES buffer (100 mM PIPES [pH 6.9], 1 mM MgCl2) for 5 min, and then washed twice in PIPES buffer. Cytoskeletons were fixed overnight in cold methanol at -20°C. For whole cells, slides were fixed for 5 min in 3% paraformaldehyde and neutralised for 10 min in 100mM Glycine, then permeabilized in 0.2% Triton X100 for 10 min. After two washes in PBS, whole cells and cytoskeletons were blocked in 1% bovine serum albumin (BSA) for 30 min. Cells were probed with anti-PFR2 (2E10B7) 1:2000, anti-HA (Sigma) 1:200, anti-mCherry (Gene Tex) 1:1000, anti-YL1/2 (Millipore) 1:1500 and anti-mNeonGreen (Chromotek) 1:1000 in 1% BSA overnight at 4°C. Slides were washed in 1% BSA before incubation with anti-IgG specific secondary antibodies conjugated to Alexa Fluor 488 (1:4000) or Alexa Fluor 546 (1:2000) (Invitrogen) for 1 h. Slides were washed in PBS, and DNA was stained with DAPI and mounted with Slowfade (Molecular Probes). Images were acquired using MetaView (Universal Imaging) software, on a Zeiss Axioplan 2 microscope equipped with a Photometrics CoolSnap CCD camera (Roper Scientific). Flagellum length measurements and processing of images were completed in ImageJ (https://imagej.nih.gov/ij/).

The Western blot assay was performed as described previously (24 (link)). The anti-mNeonGreen and anti-Flag antibodies were purchased from Chromotek and Thermo, respectively.