Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A -related kinase 5

Figure 4 Presence of never in mitosis gene A-related kinase 5 decreases tubulin tyrosine ligase-like 4 activity in a phosphorylation dependent manner. Short hairpin ribonucleic acid-never in mitosis gene A-related kinase 5 and control cells transfected with enhanced yellow fluorescent protein (EYFP)-tubulin tyrosine ligase-like (TTLL) 4 or EYFP-TTLL5 were sorted in TTLL5/TTLL4⁺ and TTLL5/TTLL4^- cells by flow cytometry. A: Sorted EYFP-TTLL4 transfected cells were lysed and analyzed by Western-blot. Stable cells expressing never in mitosis gene A-related kinase 5 were used as controls; and B: The protein extracts from EYFP-mTTLL-4 or -5 positive cells were immunoprecipitated by anti-green fluorescent protein-beads for TTLL4 or TTLL5 enrichment. The bands indicated by the black arrow correspond to TTLL4 and TTLL5, respectively, and were cut, digested and submitted to mass spectrometry. NEK5: Never in mitosis gene A-related kinase 5; GFP: Green fluorescent proteins; shRNA: Short hairpin ribonucleic acid; EYFP: Enhanced yellow fluorescent protein; TTLL: Tubulin tyrosine ligase-like.