Fig 1: In vitro disruption of microtubule network by WT and mutant TUBB8. (A) Microtubule phenotypes resulting from expression of wild-type and mutated TUBB8-flag transfected in HEK293T. a-tubulin (green) showed the endogenous microtubule network, and M2-FLAG (red) detected the expression of the transgene. Microtubules assembled into a regular network in the normal group, while the abnormal group showed a mottled pattern. The bar in normal pattern indicates 15.3 µm, and the bar in the abnormal pattern indicates 15 µm. (B) The quantitative analysis of microtubule phenotypes in panel. Approximately 200 transfected cells were examined. I bar indicates standard error. (C) Western blot of overexpressions of wild-type and mutant TUBB8. NC was a blank load transfection negative control. WT was overexpressions of wild-type TUBB8 in HEK293T. M300del showed no visible protein band when detected with anti-flag antibody, while WT and A313V had comparable expression levels. When repeated with anti-Myc antibody, M300del still had a blurry band. (D) Co-IP of TUBB8 and tubulin cofactors. WT and A313V kept interacting with both TBCD and TBCA, and S176L lost binding with TBCA but maintained interaction with TBCD. M300del had no interaction with either TBCD or TBCA.