Fig. 4: α-TubK40me3 promotes microtubule polymerization in cells and in vitro.

a Immunoprecipitation by α-TubK40me3 antibody from soluble (S) and polymerized (P) tubulins and following immunoblotting with α-tubulin showed that α-TubK40me3 was preferably distributed on polymerized but not soluble tubulins in HEK293 cells (n = 3 biological replicates). b Immunoblotting after sedimentation assay with low or high methylation level tubulins showed that α-TubK40me3 promoted microtubule formation (n = 3 biological replicates). c, d Immunoblotting and quantitative analysis showed that the ratio of P/S tubulin was decreased after SETD2 knockout in HEK293 cells. All data were shown as the mean ± s.e.m. (n = 5 biological replicates). Data were analyzed using paired two-tailed Student’s t test. *P < 0.05 versus WT. e, f Representative images and quantitative analysis at 5, 15 and 25 min from in vitro tubulin nucleation assay using 20 µM low or high methylation level tubulins showed that α-TubK40me3 highly promoted tubulin nucleation. Arrowheads indicated the microtubule nucleus. All data were shown as the mean ± s.e.m. (n = 3 biological replicates). Data were analyzed using paired two-tailed Student’s t test. *P < 0.05, ***P < 0.001 versus low methylation level. Scale bar: 5 μm. g Schematic of the observation for in vitro microtubule reconstitution by TIRF microscopy. The red part represents the GMPCPP-stabilized microtubule seeds and the green part indicates the dynamic microtubules growing from the seeds. h Kymograph of dynamic microtubules growing from seeds by time-lapse imaging with TIRF microscopy (left). Schematic diagram for the parameters of microtubule dynamics (right). i Quantitative analysis of microtubule dynamics using 20 µM low or high methylation level tubulins showed that α-TubK40me3 slightly reduced the catastrophe frequency, which may lead to increased maximum length of microtubules. All data were shown as the mean ± s.e.m. (n = 3 biological replicates, low methylation level: 93 microtubules; high methylation level: 108 microtubules). Data were analyzed using paired two-tailed Student’s t test. *P < 0.05 versus the low methylation level. Source data are provided as a Source Data file.