Figure 4. Agbl5 deficiency increases protein glutamylation in cerebellum.

(a) Schematic representation of Agbl5-KO allele. A region spanning exons 5, 6 and 7, which encodes ¾ of the entire carboxypeptidase domain of CCP5, is replaced by a selection cassette. (b) Quantitative real-time PCR using a probe spanning exon 11 and 12 shows that CCP5 RNA levels in the tissues examined are greatly reduced in heterozygous animals compared to that of wild-type littermates, and are barely detectable in the homozygous mutants. RNA levels were normalized to internal GAPDH levels and compared with the values of wild-type testis. Bars are mean ± SEM (error bars) of determinations of three animals. (c) Glutamylation or polyglutamylation levels in cerebellar lysates from wild-type (WT), pcd^3J−/− and Agbl5-KO mice are monitored by immunoblotting for GT335, B3, and polyE immunoreactivities, and (d) quantitatively analyzed using ImageStudio with normalization to α-tubulin levels. The bars represent the mean ± SEM of 3 animals of each genotype. GT335 immunoreactivity is significantly elevated in both pcd and Agbl5-KO mice compared to that of wild-type (p < 0.05), whereas B3 signal is only significantly increased in Agbl5-KO mice (p < 0.05) (Student’s t test). PolyE immunoreactivity is significantly increased in pcd cerebellum, but unaltered in Agbl5-KO cerebellum.