γ-tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies

Abstract

Monoclonal antibodies were prepared against conserved synthetic peptide from the C‐terminus of the γ‐tubulin and their specificity was confirmed by immunoblotting, competitive enzyme‐linked immunosorbent assay (ELISA) and immunofluorescence. The antibodies decorated interphase centrosomes as well as half‐spindles and midbodies in mitotic cells of various origin. The prepared antibodies were used to study the γ‐tubulin distribution in nocodazole and taxol‐treated cells. In the cells recovering from the nocodazole treatment, γ‐tubulin was found in centers of all microtubule asters. Examination of relative location of γ‐tubulin and microtubule asters in taxol‐treated mitotic cells 3T3, HeLa and PtK2 revealed that the number of taxol‐induced microtubule asters exceeded the number of γ‐tubulin‐positive spots. The γ‐tubulin was often found in the periphery of microtubule asters. Centrosomal phosphoprotein epitope detected by MPM‐2 antibody colocalized with γ‐tubulin in taxol‐treated mitotic cells. The presented data suggest that taxol‐induced microtubule asters are in vivo nucleated independently of γ‐tubulin, and other minus‐end nucleator(s) are necessary for formation of such asters. Alternatively, γ‐tubulin is present in subthreshold amounts undetectable by immunofluorescence. © 1996 Wiley‐Liss, Inc.

Microtubule Nucleation in Mouse Bone Marrow–Derived Mast Cells Is Regulated by the Concerted Action of GIT1/βPIX Proteins and Calcium

“…The following anti-peptide Abs prepared to human g-tubulin were used: mouse mAb TU-31 (IgG2b) and mAb TU-30 (IgG1) to the sequence 434-449 (14) and mAb GTU 88 (IgG1; Sigma, cat. no.…”

“…Supernatant was incubated with anti-peptide mAb TU-31 to g-tubulin immobilized on 5 ml Protein A beads overnight at 4˚C. After extensive washing in TBST (10 mM Tris-HCl [pH 7.4] 150 mM NaCl, 0.05% Tween 20), immobilized protein A with bound proteins was loaded into a 10-ml (7 3 1.5 cm) column, washed with 70 ml TBST, and eluted with peptide used for immunization (14) at a concentration of 200 mg/ml in TBST. From the 0.5-ml fractions collected, 1-ml aliquots were spotted onto nitrocellulose and probed with anti-P-Tyr mAb conjugated with HRP (dilution 1:30,000), followed by chemiluminescent detection of bound Ab.…”

STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

“…A cysteine had been added to the N terminus of the peptide to allow oriented covalent coupling to the carrier proteins, maleimide-activated keyhole limpet hemocyanin, or BSA (Imject Activated Immunogen Conjugation Kit, Pierce, Rockford, IL), according to the manufacturer's directions. BALB/c mice were immunized with the peptide-keyhole limpet hemocyanin conjugate, and sera were monitored for Ab activity by ELISA on peptide-BSA conjugate as described (23). Fusion of splenocytes with mouse myeloma cells Sp2/0, screening by ELISA, cloning and production of ascitic fluids in BALB/c mice have been described previously (24).…”

Plant γ-Tubulin Interacts with αβ-Tubulin Dimers and Forms Membrane-Associated Complexes

“…␥-Tubulin was detected with the mouse monoclonal antibody TU-31 (IgG2b) (Novakova et al, 1996) prepared against the conserved 16-amino acid peptide EYHAATRPDYISWGTQ corresponding to the human ␥-tubulin sequence 434 to 449. To generate rabbit polyclonal antibody AthTU against the 14-amino acid peptide EYKACESPD-YIKWG corresponding to the Arabidopsis ␥-tubulin sequence 437 to 450, peptide was coupled to keyhole limpet hemocyanin and antibodies were purified on protein A or affinity purified on peptide-coupled Sulfo-Link beads (Pierce).…”