Murphy SM , Urbani L , Stearns T .

	Figure 5. GCP2 and GCP3 are localized to the centrosome. (A) CHO cells were fixed and co-stained with anti-hGCP2 and either anti–α-tubulin or anti–γ-tubulin antibodies as indicated. (B) CHO cells were fixed and co-stained with anti-hGCP3 and either anti–α-tubulin or anti–γ-tubulin as indicated. Examples of cells in interphase and mitosis are shown.
	Figure 7. GCP2 and GCP3 are components of the cytoplasmic γ-tubulin complex. Epitope-tagged γ-tubulin was immunoprecipitated from cytoplasmic extracts of the indicated cell lines, with the indicated antibody in the presence or absence of competing antigenic peptide. Immunoprecipitates were immunoblotted with either anti–γ-tubulin, anti-hGCP2, or anti-hGCP3 antibodies. The positions of the 45- and 97-kD molecular weight markers are indicated.
	Figure 9. Epitope-tagged hGCP3 localizes to the centrosome and incorporates into the cytoplasmic γ-tubulin complex. (A) A31 cells stably expressing hGCP3MycHis were fixed and co-stained with anti-Myc and either anti–α-tubulin or anti–γ-tubulin, as indicated. Examples of both mitotic and interphase cells are shown. (B) Cell lysates made from A31-hGCP3MycHis were run on a sucrose gradient and fractions from this gradient were immunoblotted with anti-hGCP3 and anti-Myc antibodies. Note that epitope-tagged hGCP3 migrates more slowly than endogenous GCP3. (C) Cell lysates made from A31 or A31-hGCP3MycHis cells were subjected to immunoprecipitation with anti-Myc antibodies in the presence or absence of competing antigenic peptide. The resulting immunoprecipitates were analyzed on 7.5% SDS–polyacrylamide gels and immunoblotted with anti-hGCP2, anti-hGCP3, and anti–γ-tubulin as indicated. The positions of the 97- and 45-kD molecular weight markers are indicated.
	Figure 6. GCP2 and GCP3 cosediment with γ-tubulin in sucrose gradients. The sedimentation velocity of cytoplasmic γ-tubulin, GCP2, and GCP3 from human 293 cells was examined by sucrose gradient velocity sedimentation. Gradient fractions were immunoblotted with either anti–γ-tubulin, anti-hGCP2, or anti-hGCP3 antibodies. The position of thyroglobulin (19S) in a gradient run in parallel is indicated, as is the migration of the 45- and 97-kD molecular weight markers.
	Figure 10. GCP2 and GCP3 associate with microtubules. Equal amounts of cytoplasmic lysate from A31 cells were incubated with varying concentrations of taxol-stabilized microtubules (0– 500 pM microtubules). The highest concentration of microtubules was approximately equal to the predicted concentration of γ-tubulin complexes in these lysates. The samples were centrifuged to sediment microtubules and associated proteins. The pellets were then stained with Coomassie blue to visualize α- and β-tubulin, and immunoblotted with anti-hGCP2, anti-hGCP3, and anti–γ-tubulin antibodies. The positions of the 97- and 45-kD molecular weight markers are indicated.
	Figure 2. The composition of the mouse γ-tubulin complex. A31 (wt), and A31-derived cell lines expressing γ-tubulinMyc, γ-tubulinMycHis, and γ-tubulinHAHis were metabolically labeled with [35S]methionine and [35S]cysteine. Cytoplasmic extracts from these cells were sedimented through sucrose gradients and the epitope-tagged γ-tubulins were immunoprecipitated from fractions containing the γ-tubulin complex with the indicated antibody in the presence (+) or absence (−) of competing antigenic peptide. The resulting immunoprecipitates were analyzed on 7.5% SDS–polyacrylamide gel and visualized by fluorography. Molecular weight markers are indicated on the left. The arrows and numbers on the right indicate the position and calculated molecular weight of proteins that specifically coimmunoprecipitate with γ-tubulin. The position of the epitope-tagged γ-tubulins is indicated by the asterisk. The migration of the 100-, 101-kD doublet is altered when the anti-Myc antibody is used because the heavy chains of this antibody migrate at ∼100 kD (see Materials and Methods).
	Figure 3. There are multiple γ-tubulin molecules in the γ-tubulin complex. Epitope-tagged γ-tubulin was immunoprecipitated from cytoplasmic extracts of the indicated cell lines in the presence and absence of antigenic peptide. These immunoprecipitates were analyzed on a 7.5% SDS–polyacrylamide gel, transferred to nitrocellulose, and then immunoblotted with anti–γ-tubulin antibodies. The migration of a 45-kD molecular weight marker is indicated.
	Figure 4. hGCP3, Spc98p, hGCP2, and Spc97p are related proteins. (A) Pair-wise comparison of hGCP2, Spc97p, hGCP3, and Spc98p. The numbers represent the percent similarity using the following groupings of amino acids: N and Q; R and K; S and T; D and E; Y, F, and W; I, L, M, and V; H; P; C; G; and A. (B) A schematic diagram of the hGCP2, Spc97p, hGCP3, and Spc98p proteins. The boxes representing the proteins are drawn to scale, with the dashed segments representing gaps of 10 amino acids or more that were introduced to align the proteins. The shaded regions represent regions that show significant homology. (C) The amino acid sequences of the shaded regions of homology indicated in (B). Amino acid positions that are either identical (black shading) or similar (gray) in at least three of the proteins are indicated. These sequence data are available from GenBank/ EMBL/DDBJ under accession numbers AF042379 (hGCP2) and AF042378 (hGCP3).
	Figure 8. hGCP2 and hGCP3 migrate with the same electrophoretic mobility as the 100-, 101-kD doublet of the γ-tubulin complex. hGCP2 and hGCP3 were produced in insect cells using the baculovirus system. hGCP2 and hGCP3 were purified as insoluble proteins and analyzed on a 7.5% SDS–polyacrylamide gel, as follows: MW, molecular weight markers; 1, hGCP2; 2, hGCP3; and 3, hGCP2 plus hGCP3. The gel was stained with Coomassie blue to visualize the proteins. The molecular weights of the markers are indicated.

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