Fig. 2
From: CCP5 and CCP6 retain CP110 and negatively regulate ciliogenesis
CCP5 suppresses ciliogenesis initiation independently of its enzyme activity. A A schematic representation shows the enzymes involved in tubulin polyglutamylation and that antibodies GT335 and polyE recognize the branch point glutamate and > 3 glutamate residues in chain respectively. B When porcine tubulin was incubated with the lysate of HEK293T cells transfected with myc-tagged LacZ, CCP5 or its mutants, CCP5 reduced the GT335 immunoreactivity compared with LacZ, indicative of its deglutamylation activity, while the CCP5 mutants did not alter the GT335 immunoreactivity, indicative of enzymatic inactivity. C After 24-h serum starvation, hTERT-RPE1 cells transfected with myc-tagged LacZ, CCP5, or CCP5 mutants CCP5V251G, CCP5D295N, or CCP5R302G were immunostained with myc-tag (green) and ARL13B (red) and nuclei visualized with DAPI (blue). D Quantification of the ciliation in myc-positive cells (3 independent experiments; at least 30 cells analyzed per experimental condition, Additional file 2) revealed that similar to the wild-type CCP5, overexpression of the enzymatically inactive CCP5 mutants also suppressed cilia formation. Error bars represent s.d. ∗  ∗ , P < 0.01, Student’s t test. Scale bars: 10 µm