Ary antibody for 1 h, washed three times in PBS, and mounted

Ary antibody for 1 h, washed three times in PBS, and mounted with Prolong Gold. Images were obtained on an inverted epifluorescence microscope Nikon TE2000E, equipped with 60X 1.40 NA objective and a Photometrics CoolSnap HQ camera.Fab fragment preparation and microtubule decorationFab fragments of 6-11B-1 antibodies were purchase BTZ043 generated using the Fab K162 chemical information micro-preparation kit (Pierce). In brief, 6-11B-1 IgG was digested with immobilized papain at 37uC for 6 h with end-overend mixing. The digested products were eluted by low speed centrifugation and the Fc fragments were bound to protein A beads at room temperature for 20 min. Fab fragments were collected by low speed spin and concentrated. Untreated microtubules were incubated with GST-KHC motor domain (KR01, Cytoskeleton), whereas acetylated or deacetylated microtubules were incubated with 6-11B-1 Fab fragment at a 1:2 ratio for 1 h at room temperature. Excess motors and Fab fragments were removed by centrifugation of microtubules through a glycerol cushion (BRB80 containing 60 glycerol and 20 mM taxol) in a TLA100 rotor at 90,000 rpm for 10 min at 35uC. Fab-decorated microtubules were resuspended in BRB80 containing 20 mM taxol.Enzyme purificationHis-tagged human SIRT2 protein was bacterially expressed in BL21 (DE3) cells by inducing with 1 mM IPTG (isopropyl-b-Dthiogalactopyranoside) at 37uC for 3 h and purified under native conditions at 4uC by Ni-NTA (Qiagen) as described [26]. GSTtagged human MEC-17 was bacterially expressed in Rosetta2 cells, adsorbed to Glutathione Sepharose beads (GE Healthcare Biosciences), and eluted with in 50 mM Tris-HCl pH-8.0, 0.2 mM EDTA, 10 mM reduced glutathione as described. Purified proteins were dialyzed against dialysis buffer (20 mM Tris-HCl pH-8.0, 0.2 mM DTT) overnight at 4uC, mixed with 10 glycerol, and snap frozen in liquid nitrogen prior to storage at 280uC.Electron Microscopy and 3D ReconstructionSamples were prepared for negative stain electron microscopy using 0.75 solution of uranyl formate and conventional negative staining protocols [46]. For cryo-EM, 2 ml of the microtubule samples were applied on glow-discharged Quantifoil R2/2 200 holey carbon grids and vitrified using a Vitrobot Mark IV (FEI Co.). Vitrified 1081537 specimen was imaged on a Tecnai F20 Transmission Electron Microscope (FEI Co.) equipped with a field emission gun and operated at 200 kV. Images were recorded at a magnification of 66,964x on a Gatan US4000 CCD camera at a ,2 mm defocus. The pixel size of images acquired under these ?conditions is 2.24 A. Micrographs were screened for helical, 15-protofilament microtubules using the PHOELIX software package [47]. For 3D reconstructions of microtubule-Fab complexes, we selected filaments with strong signal at the 1/8 nm layerline in the 2D power spectra, indicative of high levels of Fab decoration. For the 3D reconstruction of the acetylated microtubule in complex with Fab, we selected and averaged layer-line data from 42 near and far sides. For the 3D recontruction of deacetylated with attached Fab and control microtubules, we selected and averaged layer-line data from 10 near and far sides.Tubulin modification and polymerizationPurified bovine brain tubulin was purchased from Cytoskeleton (TL238). Acetylated tubulin was generated by incubating purified bovine brain tubulin with recombinant GST-MEC-17 in the presence of 10 mM Acetyl coenzyme A (Sigma A2056) for 2 h at 28uC with constant agitation. Deacetylated tubulin was prepared by i.Ary antibody for 1 h, washed three times in PBS, and mounted with Prolong Gold. Images were obtained on an inverted epifluorescence microscope Nikon TE2000E, equipped with 60X 1.40 NA objective and a Photometrics CoolSnap HQ camera.Fab fragment preparation and microtubule decorationFab fragments of 6-11B-1 antibodies were generated using the Fab micro-preparation kit (Pierce). In brief, 6-11B-1 IgG was digested with immobilized papain at 37uC for 6 h with end-overend mixing. The digested products were eluted by low speed centrifugation and the Fc fragments were bound to protein A beads at room temperature for 20 min. Fab fragments were collected by low speed spin and concentrated. Untreated microtubules were incubated with GST-KHC motor domain (KR01, Cytoskeleton), whereas acetylated or deacetylated microtubules were incubated with 6-11B-1 Fab fragment at a 1:2 ratio for 1 h at room temperature. Excess motors and Fab fragments were removed by centrifugation of microtubules through a glycerol cushion (BRB80 containing 60 glycerol and 20 mM taxol) in a TLA100 rotor at 90,000 rpm for 10 min at 35uC. Fab-decorated microtubules were resuspended in BRB80 containing 20 mM taxol.Enzyme purificationHis-tagged human SIRT2 protein was bacterially expressed in BL21 (DE3) cells by inducing with 1 mM IPTG (isopropyl-b-Dthiogalactopyranoside) at 37uC for 3 h and purified under native conditions at 4uC by Ni-NTA (Qiagen) as described [26]. GSTtagged human MEC-17 was bacterially expressed in Rosetta2 cells, adsorbed to Glutathione Sepharose beads (GE Healthcare Biosciences), and eluted with in 50 mM Tris-HCl pH-8.0, 0.2 mM EDTA, 10 mM reduced glutathione as described. Purified proteins were dialyzed against dialysis buffer (20 mM Tris-HCl pH-8.0, 0.2 mM DTT) overnight at 4uC, mixed with 10 glycerol, and snap frozen in liquid nitrogen prior to storage at 280uC.Electron Microscopy and 3D ReconstructionSamples were prepared for negative stain electron microscopy using 0.75 solution of uranyl formate and conventional negative staining protocols [46]. For cryo-EM, 2 ml of the microtubule samples were applied on glow-discharged Quantifoil R2/2 200 holey carbon grids and vitrified using a Vitrobot Mark IV (FEI Co.). Vitrified 1081537 specimen was imaged on a Tecnai F20 Transmission Electron Microscope (FEI Co.) equipped with a field emission gun and operated at 200 kV. Images were recorded at a magnification of 66,964x on a Gatan US4000 CCD camera at a ,2 mm defocus. The pixel size of images acquired under these ?conditions is 2.24 A. Micrographs were screened for helical, 15-protofilament microtubules using the PHOELIX software package [47]. For 3D reconstructions of microtubule-Fab complexes, we selected filaments with strong signal at the 1/8 nm layerline in the 2D power spectra, indicative of high levels of Fab decoration. For the 3D reconstruction of the acetylated microtubule in complex with Fab, we selected and averaged layer-line data from 42 near and far sides. For the 3D recontruction of deacetylated with attached Fab and control microtubules, we selected and averaged layer-line data from 10 near and far sides.Tubulin modification and polymerizationPurified bovine brain tubulin was purchased from Cytoskeleton (TL238). Acetylated tubulin was generated by incubating purified bovine brain tubulin with recombinant GST-MEC-17 in the presence of 10 mM Acetyl coenzyme A (Sigma A2056) for 2 h at 28uC with constant agitation. Deacetylated tubulin was prepared by i.

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