Fig. 6 ∣. β-Tail glutamylation by TTLL7 stimulates TTLL6.

a, Representative images from 44 and 56 MTs analyzed for unmodified and glutamylated MTs, respectively, from two independent experiments showing Atto488–TTLL6 recruitment to unmodified and glutamylated MTs. Unmodified and glutamylated MTs are outlined in orange and blue, respectively. Average number of glutamates added to α-tubulin and β-tubulin is indicated as α + <n^E>, β + <n^E>, scale bar, 5 μm. For LC–MS spectra of MTs used in assays, see Supplementary Fig. 3. b, Normalized measurements of Atto488–TTLL6 recruitment to unmodified and glutamylated MTs; n = 44 and 56 MTs, respectively, from two independent experiments. Data are presented as mean ± s.d. ****P < 0.0001 as determined by two-tailed Mann–Whitney test. c, Representative images from 42 and 47 MTs analyzed for unmodified and glutamylated MTs, respectively, from two independent experiments showing Atto488–TTLL6ΔMTBH1-2 recruitment to unmodified and glutamylated MTs. Unmodified and glutamylated MTs are outlined in orange and blue, respectively. Average number of glutamates added to α-tubulin and β-tubulin is indicated as α + <n^E>, β + <n^E>; scale bar, 5 μm. d, Normalized measurements of Atto488–TTLL6ΔMTBH1-2 recruitment to unmodified and glutamylated MTs; n = 42 and 47 MTs, respectively, from two independent experiments. Data are presented as mean ± s.d.; NS by two-tailed Mann–Whitney test. e, Representative western blot images from one of four independent experiments showing TTLL6-mediated glutamylation of MTs glutamylated on their β-tails by TTLL7. Polyglutamylation was monitored on α-tubulin and β-tubulin using an antibodys that specifically detects polyglutamate chains (polyE); branched glutamates were monitored using the GT335 antibody (Methods). f–h, Quantification of monoglutamylation on β-tubulin (f), polyglutamylation on α-tubulin (g) and polyglutamylation on β-tubulin (h) as a function of reaction time. Modifications detected with antibodies are listed in e. n = 4 independent experiments. Error bars, s.d. *P < 0.05 and NS as determined by two-tailed t test.