Flagellar isolation and flagellar length measurement

A pH shock method was used for deflagellation followed by flagellar isolation (Craige et al., 2013; Zhu et al., 2017b). In brief, cells were deflagellated by pH shock followed by sucrose gradient centrifugation for further purification of the detached flagella. The isolated flagella were suspended in HMDEK buffer (50 mM HEPES, pH 7.2, 5 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT, 25 mM KCl) containing protease inhibitor cocktail (mini-complete EDTA-free, Roche), flash frozen in liquid nitrogen and finally stored at −80°C until use. For flagellar length measurement or phenotypic examination, cells were fixed by 1% glutaraldehyde and imaged under the Zeiss Axio Observer Z1 differential interference contrast (DIC) microscope (Carl Zeiss) with a CCD camera (QuantEM512SC, Photometric).