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 1 Never in mitosis gene A-related kinase 5 interacts with tubulin tyrosine ligase-like 4. A: Yeast colonies grew in selective medium SD-WLAH with aureobasidin and X-α-Gal. In addition to the reporter gene Auri-C we also activated the genes MEL1, ADE2 and His3 as indicators of interaction between the proteins. Controls of the yeast two-hybrid assay are presented in Supplementary Figure 3; B: Stable cells expressing never in mitosis gene A-related kinase 5 (NEK5)-FLAG were induced with 2 μg/mL of tetracycline and fractionated in cytoplasm and mitochondria. 1 µg of protein was used to immunoprecipitate NEK5 using an anti-FLAG resin, after elution the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. Tubulin tyrosine ligase-like (TTLL) 4 was co-immunoprecipitated with NEK5 as assessed with anti-TTLL4 antibody. Controls of cell fractioning are presented in Supplementary Figure 5; and C: NEK5 interacts with TTLL4, TTLL5 and TTLL7 by immunoprecipitation. HEK293T were transfected with EYFP-TTLL4, EYFP, TTLL5 and EYFP-TTLL7. Endogenous NEK5 was immunoprecipitated using anti-NEK5 antibody. Immunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The identification of TTLLs was assessed by an anti-green fluorescent protein antibody to confirm interaction between the proteins. NEK5: Never in mitosis gene A-related kinase 5; GFP: Green fluorescent proteins; EYFP: Enhanced yellow fluorescent protein; TTLL: Tubulin tyrosine ligase-like.