Porcine brain tubulin

Microtubules were prepared by polymerizing 5 mg/ml porcine brain tubulin (Cytoskeleton, Denver, CO) in polymerization buffer (80 mM PIPES, pH 6.8, 1 mM EGTA, 4 mM MgCl2, 2 mM GTP, 9% dimethyl sulfoxide) for 30 min at 37°C. Paclitaxel was added at 250 μM before further incubation of 30 min at 37°C. The polymerized microtubules were then incubated at room temperature for several hours before use. Microtubules (2.5 uM) diluted in BRB80 were absorbed for 1 min to carbon-coated, glow-discharged grids. 4 μL of the Astrin 324–693 complex (~250 nM complex in BRB80) was then added to the microtubules and after a short incubation the grid was blotted and negatively stained with 1% Uranyl Acetate. Single particles of both Astrin 324–693 and Astrin 465–693 complexes (~25 nM in Column Buffer) were incubated shortly on a glow discharged carbon-coated grids and stained with 1% Uranyl Acetate and Uranyl Formate respectively. Images were acquired on a FEI Tecnai TF20 200kV FEG Transmission Electron Microscope (TEM) (FEI, Hillsboro, Oregon), using a 4K × 4K Teitz (Gauting, Germany) camera.

Sagittal sections from paraffin embedded mouse brain were cut at a thickness of 5 μm. After deparaffinization, matched sections were heat retrieved in 0.01M sodium citrate buffer (pH 6.0) containing 0.05% Tween-20 (Sigma, St. Louis, MO) for 20 min at 98°C. After blocking with 10% normal horse serum (Vector Labs, Burlingame, CA) in PBS, sections were incubated overnight at 4°C with antibodies to Calbindin D-28-K (1:1,000, EMD Millipore, USA), Tbr2 (1:400, Abcam), or GT335 monoclonal antibody (1:1,000, Adipogen, San Diego, CA, USA) or GT335 antibody pre-absorbed with porcine brain tubulin (Cytoskeleton Inc, Denver, CO), then incubated for 1 h at room temperature with Alexa 488-labeled donkey anti-mouse or anti-rabbit antibody (1:200, Invitrogen, San Diego, CA). Sections were counterstained with DAPI (Invitrogen, San Diego, CA).

Common chemicals were purchased from Fisher Scientific and Sigma-Aldrich. Porcine brain tubulin, GTP and paclitaxel were purchased from Cytoskeleton, Inc. 99.8% D₂O, ¹⁵NH₄Cl and U-¹³C₆ glucose, 2-¹³C glucose, 1,6-¹³C glucose, U-¹³C₆,D₇ glucose were purchased from Cambridge Laboratories, Inc. EDTA-free protease inhibitor tablets were obtained from Roche. Chromatography columns were purchased from GE Healthcare. The 400 mesh copper grids coated with formvar and stabilized with evaporated carbon films were purchased from Electron Microscopy Science.
The human KIF5B motor domain construct (amino acids 1–349) was prepared using the pET28b vector fused with a His₆-SMT3 Tag at the N-terminus (in the form of His₆-SMT3-KIF5B). The His₆-SMT3-KIF5B was transformed into Escherichia coli BL21(DE3) cells. The His₆-Ulp1 was expressed and purified from the His₆-Ulp1 protease construct kindly provided by Dr. Christopher Lima from HHMI and Memorial Sloan Kettering Cancer Center, New York, NY 10065.