To localize γ-tubulin in vivo, log phase cells from strain tG2N-HA or cTTMG-HA were processed for immunofluorescent labeling as described (Gaertig et al., 1995 (
link)). HA antibody (16B12) was used at a 1:1,000 dilution. Secondary antibodies were goat anti–mouse FITC (Sigma-Aldrich) at a 1:200 dilution or Alexa fluor 568 goat anti–mouse IgG (Molecular Probes) at a 1:500 dilution. DAPI (4′, 6′-diamidino-2-phenylindole) was used for DNA staining at 100 ng/ml. To study the effects of γ-tubulin depletion on BBs,
GTU1 knockout heterokaryon progeny or cTTMG-HA cells in CdCl
2-free SPP medium were stained with anti-centrin monoclonal or polyclonal antibodies, provided by Dr. Jeffrey L. Salisbury (Mayo Clinic Foundation, Rochester, NY). Anti-glutamic acid polyclonal antibody (R-polyE), a rabbit antibody raised commercially (Alpha Diagnostic International) against a Cys (Glu)
9 peptide coupled to keyhole limpet hemocyanin, was used to stain BBs and cilia. MT structures were stained with anti–α-tubulin antibodies (DM1A; Sigma-Aldrich). All antibodies were used at a 1:1,000 dilution. Anti-TFKBP12 polyclonal antibody, provided by Dr. Osamu Numata (University of Tsukuba, Ibaraki, Japan) was used to stain the TFKBP12 (
Tetrahymena thermophila FK506 binding protein of 12 kD) at a 1:250 dilution. Secondary antibodies were goat anti–mouse FITC (Zymed Laboratories, Inc.), goat anti–rabbit rhodamine (Zymed), and goat anti–rabbit FITC (Sigma) at 1:200 dilutions. To stain kinetodesmal fibers (KF, also known as striated rootlets), 2–5 × 10
5 depleted cells were placed on ice for 5 min, fixed in 35% EtOH/0.12% Triton X-100 on ice for 30 min, washed with cold TBS twice (Nelsen et al., 1994 (
link)), and stained with mAb FI-5D8 (Jerka-Dziadosz et al., 1995 (
link)) provided by Dr. Joseph Frankel (University of Iowa, Iowa City, IA). Secondary antibodies, Alexa fluor 568 goat anti–mouse IgG (Molecular Probes) and goat anti–rabbit FITC (Sigma-Aldrich), were used at a 1:500 or 1:250 dilution, respectively. Four to eight stacked images of cell were obtained using a Leica TCS SP confocal microscope (PL Apo 100× lens, N.A. 1.4, and 1.8× zoom).
Shang Y., Li B, & Gorovsky M.A. (2002). Tetrahymena thermophila contains a conventional γ-tubulin that is differentially required for the maintenance of different microtubule-organizing centers. The Journal of Cell Biology, 158(7), 1195-1206.