Expression and Characterization of Tubulin Mutants

Human KL1-expression vector was sourced commercially (RC220944; OriGene Technologies, Rockville, MD, USA) and the KL1 protein was expressed in a pEGFP-C1 vector (Clontech, Mountain View, CA, USA) (GFP-KL1) as previously reported [20 (link)]. Mouse α-TAT1 expression vector was also purchased commercially (MR206707; OriGene Technologies) and flag-tagged α-TAT1 was expressed from a pCMV6-entry vector. The expression vector for rat katanin (pEGFP-C1-p60 katanin) was kind gift of Dr. PW. Baas, Drexel University, Philadelphia, PA. The human α-tubulin construct [20 (link)] was N-terminally GFP-tagged and expressed in pEGFP-C1 (GFP-tubulin(wt)). The tubulin K40R mutation was introduced using a GeneArt Site-Directed Mutagenesis PLUS kit (Life Technologies, Carlsbad, CA, USA) (GFP-tubulin(K40R)) and the structure of the construct was confirmed by sequencing.