Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 1. The evolutionary conserved Thr72 in human TPX2 is phosphory- lated by Cdk1 and Cdk2 in vitro. A, comparative alignment of part of human TPX2 sequence (amino acids 41 to 80) with corresponding sequences from other species (mouse, rat, and frog) using DNAMAN software (Lynnon Corpo- ration). The sequence alignment shows that Thr72 in human TPX2 is con- served in all other species. B, schematic diagram of the experimental protocol for mass spectrometry analysis (LC-MS/MS) using mitotic HeLa cells. HeLa cells were synchronized at M phase by nocodazole treatment (100 ng/ml) for 16 h and released for 30 min after nocodazole washout. Endogenous TPX2 was immunoprecipitated from 10 mg of total protein using pan-TPX2 Abs (clone 184). IP sample was run on SDS-PAGE and after Coomassie Blue stain- ing, the band with the matching size to TPX2 (confirmed by Western blotting with TPX2 Abs, not shown) was cut out and sent for LC-MS/MS analysis. The gray asterisk on the spectra of the phosphopeptide containing Thr72 indicate the identified matched fragment ions on mass spectrometry. C, phosphoryl- ation sites identified by mass spectrometry analysis on endogenous TPX2 immunoprecipitated from nocodazole-synchronized mitotic HeLa cells in regards to the known TPX2 domains. All these sites have been identified previously (17, 19–32) but not confirmed and analyzed. Thr72 is the first vali- dated and functionally characterized phosphorylation site in human TPX2 (this study). D, in vitro kinase assay using purified Cdk1/2 proteins and phos- phospecific Thr72 TPX2 Abs. Purified GST fusion protein TPX2 WT, GST-TPX2- T72A, or GST-TPX2-T72E was incubated with each active Cdk-cyclin complex inthepresenceof1mMcoldATP.Allkinasereactionswerestoppedbyadding 2 SDS sample buffer and the samples were run on SDS-polyacrylamide gel electrophoresis followed by Western blot detection using the indicated antibodies.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: In Vitro, Sequencing, Software, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, SDS Page, Staining, Western Blot, Phospho-proteomics, Kinase Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 2. Thr(P)72 TPX2 antibodies are specific in Western blot for TPX2 phosphorylated at Thr72 in vivo. A, specificity of Thr(P)72 TPX2 for TPX2 proteintestedbysiRNA.HeLacellsweretransfectedwithcontrolsiRNAorone of two TPX2 siRNAs for 24 h and synchronized at M phase with nocodazole treatment (100 ng/ml). Cells were harvested and lysed with lysis buffer. Sam- ples were run on SDS-PAGE, followed by Western blotting, first probed with the Thr(P)72 TPX2 Abs, then stripped and re-probed with pan-TPX2 (clone 184) Abs. Levels of actin were used as loading controls. B, bar graph quanti- tation for the relative expression levels of Thr(P)72 TPX2 and TPX2 in control and TPX2 siRNA-transfected cells. Each sample was compared with sample treated with control siRNA. Relative expression levels of Thr(P)72 for control siRNA,10;TPX2siRNA#1(UTR),0.3180.085;TPX2siRNA#2(Cds),0.289 0.115. Relative expression levels of TPX2, control siRNA, 1 0; TPX2 siRNA #1, 0.335 0.0074; TPX2 siRNA #2, 0.304 0.091 (mean S.E.). n 4 samples, from 4 independent experiments. Unpaired Student’s t test indicated all the results are significant. ***, p 0.001. C, specificity of Thr(P)72 TPX2 tested by the use of T72A mutant and -PPase treatment. HeLa cells were left untrans- fected, transfected with an empty GFP vector, GFP-TPX2 WT, or GFP-TPX2 T72A mutant plasmids. 24 h after transfection, cells were synchronized with nocodazole for 16 h, harvested, and lysed. TPX2 immunoprecipitation was performed in each sample with TPX2 Abs (clone 183). Where indicated, IP

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Western Blot, In Vivo, Lysis, SDS Page, Expressing, Control, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 3. In vivo TPX2 phosphorylation at Thr72 is cell cycle-dependent and peaks at M phase. A, cell cycle profiles analyzed by flow cytometry analysis to confirmcellsynchronizationateachphase.B,afterIPswithpan-TPX2Abs(clone184),WesternblotswereprobedfirstwiththeThr(P)72TPX2Absandthen,after stripping, re-probed with pan-TPX2 Abs (clone 184). The levels of -actin were used as loading controls. The levels of cyclin B1 were used as positive controls to show that synchronization at M phase worked well, as indicated by the high level of cyclin B1 in M phase compared with that of S phase or non-synchronized cells. The Western blot figures are representative of 3 independent experiments. The input blot is from the mitotic samples. C, bar graphs for quantification of relative levels of Thr72 phosphorylation are shown. Non-syn, non-synchronized cells (1 0); M, M phase cells (4.16 0.36); S, S phase cells (1.31 0.4); mean of the relative levels of Thr(P)72 TPX2/TPX2 expression S.D., n 3 independent experiments; ***, non-syn versus M phase, p 0.001; **, M versus S phase, p 0.01; NS, not significant: non-syn versus S phase, all by unpaired Student’s t test.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: In Vivo, Phospho-proteomics, Flow Cytometry, Stripping Membranes, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 4. Phosphorylation of TPX2 at Thr72 is inhibited by the Cdk inhibitor roscovitine. A, the levels of Thr(P)72 were reduced by roscovitine treatment. HeLa cells were first treated with 100 ng/ml of nocodazole for 16 h and then once synchronized, treated with dimethyl sulfoxide (as a control), or 20 and 40 M roscovitine for 30 min. Cells were harvested, lysed, and TPX2 was immunoprecipitated with pan-TPX2 Abs (clone 184). SDS-PAGE was performed and followed by Western blotting with Thr(P)72 and pan-TPX2 Abs (clone 184). B, cyclin B1 levels were also used to confirm that roscovitine-treated cells had remained in mitosis. The levels of p-Cdk/MAPK substrates (PX(S*/T*)P or (S*/T*)PX(R/K) motif) were also used to confirm the effectiveness of the treatment. Actin levels were used as loading control.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Phospho-proteomics, Control, Immunoprecipitation, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 5. Localization of Thr(P)72 TPX2 in HeLa and 293 cells. Mitotic (A and B) and interphase (C) HeLa cells were stained with Abs directed against Thr(P)72 TPX2, TPX2, and tubulin or with the Thr(P)72 Abs pre-absorbed with blocking peptide at different ratios. A, in mitotic cells, TPX2 phosphorylated at Thr72 is localized in the cytosol and does not strictly associate with the mitotic spindle. B, HeLa cells stained with pan-TPX2 and Thr(P)72 TPX2 Abs preincubated with Thr(P)72 blocking peptides. C, during interphase, Thr(P)72 TPX2 is localized in the nucleus. Note that the expression levels of Thr(P)72 are much lower in interphase cells than in mitotic cells. Note that only the Thr(P)72 signal was blocked. D, representative photographs of mitotic 293 cells transfected with GFP-TPX2 WT (WT) and GFP-TPX2 T72A (T72A). Scatter plots show the GFP signal at microtubules relative to total GFP signal. GFP-TPX2 T72A is significantly enriched on microtubules when compared with GFP-TPX2 WT (GFP-TPX2 WT (0.26 0.01, n 29) versus GFP-TPX2 T72A (0.39 0.02, n 22), group (mean S.E.); ***, p 0.0001 by t test). Scale bar, 10 m.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Staining, Blocking Assay, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 6. Effects of GFP-TPX2 T72A on the polarity of mitotic spindles in HeLa cells with or without endogenous TPX2. A, representative photographs of mitotic HeLa cells at prometaphase and metaphase with monopolar, bipolar, and multipolar mitotic spindle poles. Scale bar, 10 m. B, Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in cells with intact levels of TPX2. C, bar graphs showing the number of cells with different mono-, bi-, or multipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. GFP-TPX2 T72A expression results in a significant increase in the percentage of cells with multipolar spindles in the presence of endogenous TPX2. ANOVA comparing the three groups shows high significance with p 0.001. Neuman-Keuls test was used to compare each group: GFP (1.49 0.47) versus T72A (12.72 2.10), p 0.001; TPX2 WT (3.36 0.40) versus T72A (12.72 2.10), p 0.001; group (mean S.E.); ***, p 0.001; NS, not significant (GFP versus TPX2). At least 100 cells for each set of experiments were used for quantification, 5 independent experiments were performed. Error bars indicate S.E. D, Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in HeLa cells co-transfected with GFP-vector, GFP-TPX2 WT, or GFP-TPX2T72AtogetherwithTPX2siRNAtargetingthe3UTRofTPX2mRNA.E,bargraphsshowingthenumberofcellswithdifferentmono-,bi-,ormultipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. Knockdown of TPX2 in GFP-transfected cells results in a significant 5.4% increase in multipolar spindles versus control cells without TPX2 depletion. GFP-TPX2 T72A expression produces an even greater 9.8 and 7.5% increase in the percentage of cells with multipolar spindles when compared with GFP/TPX2 siRNA and GFP-TPX2 WT/TPX siRNA, respectively. n 3, ANOVA test was used the compare the four groups (p 0.01). The Neuman-Keuls test was used to compare the following groups: control (with control siRNA) (2.43 0.41) versus GFP (7.94 1.5), p 0.05; GFP (7.94 1.5) versus TPX2 WT (10.13 1.2), NS; WT (10.13 1.2) versus T72A (17.67 3.2), p 0.05; GFP (7.94 1.5) versus T72A (17.67 3.2), p 0.05; group (mean S.E.); *, p 0.05; NS, not significant. n at least 500 cells for each set of experiments; 3 independent experiments were performed. Error bars indicate S.E.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Western Blot, Staining, Expressing, Transfection, Plasmid Preparation, Knockdown, Control

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly

doi: 10.1074/jbc.m114.591545

Figure Lengend Snippet: FIGURE 7. Overactivation of Aurora A and increased spindle length, a measure of Eg5 activity, in TPX2 T72A-expressing cells. A–C show 293 mitotic cells (prometaphase/metaphase) previously transfected with GFP-TPX2 WT (WT) or GFP-TPX2 T72A (T72A) expression vectors. A, representative photographs of WT- and T72A-transfected cells stained for Thr(P)288, a phosphoresidue indicative of the activity of Aurora kinase A. Dotted circles identify the poles. Scatter plots show the P-Aurora signal at centrosomes relative to total GFP signal. GFP-TPX2 T72A induces higher Aurora A activity than GFP-TPX2 WT (GFP-TPX2 WT (0.07 0.01, n 13) versus GFP-TPX2 T72A (0.14 0.03, n 18); *, p 0.05 by t test). B, representative photographs of the spindle length detected in mitotic 293 cells transfected with GFP-TPX2 WT or T72A. Scatter plots show the spindle length in both groups. T72A- expressing cells display longer spindles than WT-expressing cells (GFP-TPX2 WT (58.95 2.12, n 18) versus GFP-TPX2 T72A (66.67 2.20, n 21); **, p 0.01 by t test). C, representative images of the -tubulin signal detected in GFP-TPX2 WT and T72A-trasnfected 293 cells. No significant difference was detected between these two groups (GFP-TPX2 WT (1.93 0.41, n 15) versus GFP-TPX2 T72A (1.97 0.49, n 13); NS, non significant by t test). In all the panels: the group is the mean S.E.. Scale bar, 10 m.

Article Snippet: Antibodies—Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P)72 TPX2 (homemade, described above), Cyclin B (Abcam), 9124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 14 • APRIL 3, 2015 at H A R V A R D U N IV E R SIT Y on A pril 15, 2015 http://w w w .jbc.org/ D ow nloaded from -actin (Chemicon), TPX2 (clone 183, epitope: 150 –200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700 –749 amino acids of TPX2, Novus Biologicals), phosphoCdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Activity Assay, Expressing, Transfection, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Combined functional genome survey of therapeutic targets for hepatocellular carcinoma.

doi: 10.1158/1078-0432.CCR-09-2214

Figure Lengend Snippet: Fig. 5. Protein expression in HCC. Hematoxylin and eosin (HE) staining (original magnification, × 100) and immunoperoxidase staining (original magnifications, × 100 and × 400) of AKR1B10, HCAP-G, RRM2, and TPX2 proteins in HCC and adjacent nontumorous liver tissue. The specificity of antibodies was determined by immunoblotting of the KIM-1 cell lysate (left). N, nontumorous liver.

Article Snippet: Anti-TPX2 antibody was purchased from Novus Biologicals.

Techniques: Expressing, Staining, Immunoperoxidase Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

doi: 10.1074/jbc.m112.385674

Figure Lengend Snippet: FIGURE 2. Effects of TPX2 overexpression on the levels of -H2AX and MDC1 ionizing radiation-induced foci. A and B, overexpression of His-TPX2 or GFP-TPX2 significantly decreases phosphorylation of H2AX in HeLa (A) and MCF-7 cells (B) 1 h after ionizing radiation treatment as indicated by Western blot analysis (10 Gy) (A) and immunofluorescence microscopy (B) (left panel, 2Gy; right panel, 4Gy). A, relative quantifications of -H2AX signals from independent experiments are shown in bar charts: control IR (100.0 6.9) versus His-TPX2 IR (16.0 2.9), p 0.001, n 4 (independent experiments); group (mean S.E.), unpaired t test. Levels of actin and H2A were used as loading controls. NS, non-significant; ***, p 0.001. Error bars represent S.E. C, dose-dependent effects of GFP-TPX2 or His-TPX2 on the levels of ionizing radiation-induced -H2AX. The amount of plasmid transiently transfected per 6-cm cell culture dish is indicated. Proteins on Western blots were visualized with the indicated antibodies. TPX2 antibodies recognize endogenous and exogenous TPX2 on the same Western blot, thereby allowing comparison of absolute protein levels (see text for details). D, overexpression of His-TPX2 or GFP-TPX2 inhibits MDC1 ionizing radiation-induced focus formation in MCF-7 cells after 2 or 4 Gy (15-min recovery), respectively. Bar in B and D, 10 m. Merged images include DAPI staining (B and D). A.U., arbitrary units. Transfected cells are indicated by white frame and asterisks (B and D).

Article Snippet: The specificity of TPX2 antibody (184; Novus Biologicals) used for immunostainingwas determined by peptide competition (supplemental Fig. 5) and depletion of TPX2 by RNAi (see below).

Techniques: Over Expression, Phospho-proteomics, Western Blot, Immunofluorescence, Microscopy, Control, Plasmid Preparation, Transfection, Cell Culture, Comparison, Staining

Journal: Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

doi: 10.1074/jbc.m112.385674

Figure Lengend Snippet: FIGURE 3. TPX2 localizes to DNA double strand breaks. A and B, TPX2 partially co-localizes with -H2AX-positive ionizing radiation-induced foci after 5 Gy in LAN1 (A) and U2OS cells (B), respectively (see also supplemental Fig. 5). TPX2 is found in the nucleus and cytosol in neuroblastoma LAN1 cells and neurons (see text and supplemental Fig. 5 for details). C, TPX2 co-localizes with -H2AX at 4-hydroxytamoxifen (4-OHT)/AsiSI-induced DNA double strand breaks. U2OS cells stably expressing AsiSI-estrogen receptor were left untreated or treated with 300 nM 4-hydroxytamoxifen for 4 h and subsequently immunostained for TPX2 and -H2AX. D, TPX2 accumulates in DNA double strand break-containing laser tracks (indicated by white arrows) marked by -H2AX. U2OS cells were either mock-treated (MP) or microirradiated with a multiphoton laser (MP). A representative image shows TPX2 accumulation at 10 min after irradiation. The intensity profiles of -H2AX and TPX2 immunofluorescence signals were measured in the yellow bars perpendicular to the laser tracks. Commercially available TPX2 antibody 184 was used in all immunofluorescence images. See supplemental Fig. 5 and Fig. 5 for specificity of TPX2 184 antibody. Bars, 10 m. AU, arbitrary units.

Article Snippet: The specificity of TPX2 antibody (184; Novus Biologicals) used for immunostainingwas determined by peptide competition (supplemental Fig. 5) and depletion of TPX2 by RNAi (see below).

Techniques: Stable Transfection, Expressing, Irradiation, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

doi: 10.1074/jbc.m112.385674

Figure Lengend Snippet: FIGURE 4. TPX2 regulates the levels of ionizing radiation-dependent -H2AX and forms ionizing radiation-induced foci in G1 phase cells. A, enhanced levels of -H2AX 1 h after 10 Gy in G1 phase HeLa cells depleted of TPX2 by doxycycline-induced TPX2 miRNA expression. Note that the -H2AX augmentation in these TPX2-depleted cells is ionizing radiation-dependent. Cells were synchronized using a double thymidine block, released into fresh medium, and then used at specified time points for ionizing radiation treatment as indicated. Unirradiated cells were used for flow cytometry-based cell cycle profiling (top bar charts). Relative quantifications of -H2AX signals from independent experiments are shown in bar charts on the right: 11 h, doxycycline IR (229.0 21.0) versusdoxycyclineIR(748.639.4),p0.001;12h,doxycyclineIR(135.134.2)versusdoxycyclineIR(466.198.6),p0.05;group(meanS.E.), unpaired t test; n 3 (independent experiments); *, p 0.05; ***, p 0.001. Error bars represent S.E. Note that 11 h after release from the thymidine block TPX2-depleted cultures contain slightly more G2/M phase cells (8.66%) than control cultures (3.25%). Thus, on the Western blot, a 12 h-11 h loading hierarchy ischosentofacilitatecomparisonbetweencontrol11hversusdoxycycline12h(3.96%G2/Mcells).Althoughthecellcycleprofilebetweenthesetwosamples is highly similar, TPX2-depleted cells exhibit approximately twice the levels of -H2AX than control cells. B, U2OS cell cultures synchronized with a double thymidineblockasinA(seeflowcytometry-basedcellcycleprofilesintophistograms;NS,non-synchronizedcontrol)formTPX2ionizingradiation-inducedfoci 1 h after irradiation that partially co-localize with -H2AX during G1 phase. All images were taken under identical experimental and microscopic conditions. See text for details. C, TPX2 maintains its association with the mitotic spindle in the presence of DNA double strand breaks marked by -H2AX. Early and late mitotic figures as identified via DAPI and TPX2 staining with and without DNA damage are shown. Commercially available TPX2 antibody 184 was used in all immunofluorescence images. See supplemental Fig. 5 and Fig. 5 for specificity of TPX2 184 antibody. A.U., arbitrary units.

Article Snippet: The specificity of TPX2 antibody (184; Novus Biologicals) used for immunostainingwas determined by peptide competition (supplemental Fig. 5) and depletion of TPX2 by RNAi (see below).

Techniques: Expressing, Blocking Assay, Flow Cytometry, Control, Western Blot, Irradiation, Staining, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

doi: 10.1074/jbc.m112.385674

Figure Lengend Snippet: FIGURE 5. Regulation of -H2AX levels by TPX2 is distinct from its mitotic functions and occurs in postmitotic G0 primary neurons. A, depletion of Ndel1 does not increase -H2AX levels after ionizing radiation treatment. B, depletion of NUF2 does not increase -H2AX levels after ionizing radiation treatment. The mitotic arrest upon NUF2 depletion was confirmed with the increased levels of the mitotic marker H3S10p (see text for details). C, significant decrease in TPX2 levels in mouse primary cortical neurons co-transfected with a TPX2 shRNA-encoding construct and GFP (ratio, 5:1) as detected by immunofluorescence and confocal microscopy using commercially available TPX2 antibody 184. See supplemental Fig. 5 for the expression pattern of TPX2 in brain tissues, cellular distribution of TPX2 in primary neurons, and specificity of the TPX2 shRNA and antibody 184. D, enhanced levels of -H2AX in G0 postmitotic primary neurons co-transfected with vectors encoding TPX2 shRNA and GFP (ratio, 10:1) 1 h after 10 Gy compared with surrounding untransfected cells or cells co-transfected with vectors encoding a control shRNA and GFP (ratio, 10:1) as determined by immunofluorescence and confocal microscopy. E, decreased levels of -H2AX in G0 postmitotic primary neurons transfected with GFP-TPX2 compared with surrounding untransfected cells or cells transfected with GFP 1 h after 10 Gy as determined by immunofluorescence and confocal microscopy. Note that the -H2AX signals in control shRNA- and GFP-expressing neurons are of an intensity similar to that of untransfected surrounding cells. F and G, quantification of the relative changes in -H2AX signals in neurons in D and E expressed by the average ratio of total nuclear -H2AX signals of transfected neurons/average total nuclear -H2AX signals of non-transfected surrounding cells. F, ratio control shRNA (n 10)/non-transfected (n 196) 1.7 0.4 (S.E.) versus ratio TPX2 shRNA (n 16)/non-transfected (n 459) 6.4 0.9 (S.E.); p 0.001, unpaired ttest.G,ratioGFP(n9)/non-transfected(n37)1.00.1(S.E.)versusratioGFP-TPX2(n8)/non-transfected(n33)0.50.1(S.E.);p0.001,unpaired t test. H and I, quantification of the relative changes in -H2AX immunofluorescence signals in neurons co-transfected with vectors encoding a control or TPX2 shRNAandGFP(ratio,10:1)(H)oraGFPorGFP-TPX2construct(I)1hafter3Gy.H,ratiocontrolshRNA(n58)/non-transfected(n300)1.10.1(S.E.)versus ratio TPX2 shRNA (n 75)/non-transfected (n 330) 1.3 0.1 (S.E.); p 0.001, unpaired t test. I, ratio GFP (n 50)/non-transfected (n 297) 1.2 0.1 (S.E.) versus ratio GFP-TPX2 (n 47)/non-transfected (n 420) 0.9 0.1 (S.E.); p 0.01, unpaired t test. Values were calculated as in F and G. NS, non-significant; **, p 0.01; ***, p 0.001. Error bars represent S.E. Bars, 20 m. Ctrl., control. White arrows indicate transfected cells (C–E).

Article Snippet: The specificity of TPX2 antibody (184; Novus Biologicals) used for immunostainingwas determined by peptide competition (supplemental Fig. 5) and depletion of TPX2 by RNAi (see below).

Techniques: Marker, Transfection, shRNA, Construct, Immunofluorescence, Confocal Microscopy, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

doi: 10.1074/jbc.m112.385674

Figure Lengend Snippet: FIGURE 6. TPX2 associates with MDC1, p-ATM, NBS1, and -H2AX. A, Y2H experiment using bait-TPX2 (aa 8–747) and prey-MDC1 (aa 1683–2089 or 1809–2089). Plasmids were co-transformed as indicated. Leu/Trp selection agar plates are controls for transformation efficiency. Colonies on Ade/His/ Leu/Trp selection agar plates reveal an interaction between bait-TPX2 and prey-MDC1. Plates were incubated for 6 days. B, an ectopic C-terminal fragment ofMDC1(C-MDC1-FLAG;aa1807–2089)associateswithendogenousTPX2inHeLacellsasindicatedbyco-immunoprecipitationswithTPX2antibody184from totalcelllysate(leftpanel).EctopicGFP-TPX2alsoco-immunoprecipitateswithendogenousMDC1(rightpanel).TheinputlanefortheC-MDC1-FLAG(leftpanel) is from a shorter exposure of the same Western blot. The input lane for the endogenous MDC1 (right panel) is from a stronger exposure of the same Western blot. C, GST-TPX2 pulls down MDC1 and the positive control Aurora A from total HeLa cell lysate. For MDC1, the input is a shorter exposure of the same blot. D, co-immunoprecipitations from U2OS cell lysates with the specified antibodies (see text for details on antibodies). TPX2 was co-immunoprecipitated with MDC1 from these cells using MDC1 antibody (left panel). MDC1 also co-immunoprecipitated with TPX2 antibody 184 (left panel). TPX2 was also found in complex with NBS1 and MDC1 species that migrate slower on SDS-PAGE gels when the co-immunoprecipitations were performed with the TPX2 KiS2 antibody (right panel; see text for further details). E, TPX2 and MDC1 associate in HeLa and LAN1 cells as detected by co-immunoprecipitations with the specified antibodies from total (left and middle panels) or nuclear (right panel) lysates. TPX2 from neuroblastoma LAN1 cells (and primary neurons) migrates as a doublet on gels (see supplemental Fig. 5). TPX2 is also found in complex with p-ATM and -H2AX after ionizing radiation treatment. Beads alone or antibodies against c-Myc were used as negative controls as indicated. IP, immunoprecipitation.

Article Snippet: The specificity of TPX2 antibody (184; Novus Biologicals) used for immunostainingwas determined by peptide competition (supplemental Fig. 5) and depletion of TPX2 by RNAi (see below).

Techniques: Transformation Assay, Selection, Incubation, Western Blot, Positive Control, Immunoprecipitation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

doi: 10.1074/jbc.m112.385674

Figure Lengend Snippet: FIGURE 7. The ionizing radiation-dependent increase in -H2AX caused by TPX2 depletion is antagonized by inhibition or knockdown of MDC1 or ATM. A, an siRNA-mediated knockdown of MDC1 antagonizes the ionizing radiation-triggered -H2AX hyperamplification in HeLa cells caused by TPX2 depletion(lane3versuslane4).NotethatduringtheDNAdamageresponseTPX2depletionandMDC1depletionhaveanoppositeeffecton-H2AXlevels(lane 1 versus lane 3). B, inhibition of ATM with KU55933 antagonizes the ionizing radiation-dependent increase in -H2AX caused by depletion of TPX2. Inhibition of DNA-PK with NU7441 does not rescue this -H2AX hyperamplification phenotype. C, siRNA-mediated loss of ATM abrogates the ionizing radiation-depen- dent increase in -H2AX caused by depletion of TPX2. siRNA-mediated loss of DNA-PKcs partially decreases ATM levels (as previously described in Ref. 71) but does not rescue the -H2AX hyperamplification caused by TPX2 depletion. D, quantification of -H2AX signals in C. -H2AX signals of control siRNA-treated cells were considered as 100% and compared with the respective TPX2 siRNA-treated cells (n 3 (independent experiments); N.S., non-significant; *, p 0.05, unpaired t test, S.E.). Error bars represent S.E. All cells were treated with 10 Gy and harvested after 1-h recovery. Ctrl., control.

Article Snippet: The specificity of TPX2 antibody (184; Novus Biologicals) used for immunostainingwas determined by peptide competition (supplemental Fig. 5) and depletion of TPX2 by RNAi (see below).

Techniques: Inhibition, Knockdown, Control

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: The evolutionary conserved Thr72 in human TPX2 is phosphorylated by Cdk1 and Cdk2 in vitro. A, comparative alignment of part of human TPX2 sequence (amino acids 41 to 80) with corresponding sequences from other species (mouse, rat, and frog) using DNAMAN software (Lynnon Corporation). The sequence alignment shows that Thr72 in human TPX2 is conserved in all other species. B, schematic diagram of the experimental protocol for mass spectrometry analysis (LC-MS/MS) using mitotic HeLa cells. HeLa cells were synchronized at M phase by nocodazole treatment (100 ng/ml) for 16 h and released for 30 min after nocodazole washout. Endogenous TPX2 was immunoprecipitated from 10 mg of total protein using pan-TPX2 Abs (clone 184). IP sample was run on SDS-PAGE and after Coomassie Blue staining, the band with the matching size to TPX2 (confirmed by Western blotting with TPX2 Abs, not shown) was cut out and sent for LC-MS/MS analysis. The gray asterisk on the spectra of the phosphopeptide containing Thr72 indicate the identified matched fragment ions on mass spectrometry. C, phosphorylation sites identified by mass spectrometry analysis on endogenous TPX2 immunoprecipitated from nocodazole-synchronized mitotic HeLa cells in regards to the known TPX2 domains. All these sites have been identified previously (17, 19,–32) but not confirmed and analyzed. Thr72 is the first validated and functionally characterized phosphorylation site in human TPX2 (this study). D, in vitro kinase assay using purified Cdk1/2 proteins and phosphospecific Thr72 TPX2 Abs. Purified GST fusion protein TPX2 WT, GST-TPX2-T72A, or GST-TPX2-T72E was incubated with each active Cdk-cyclin complex in the presence of 1 mm cold ATP. All kinase reactions were stopped by adding 2× SDS sample buffer and the samples were run on SDS-polyacrylamide gel electrophoresis followed by Western blot detection using the indicated antibodies.

Article Snippet: Endogenous TPX2 was immunoprecipitated from 10 mg of total protein lysates using TPX2 Abs (clone 184, Novus Biologicals) and Protein A/G-Sepharose 4 Fast Flow beads.

Techniques: In Vitro, Sequencing, Software, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, SDS Page, Staining, Western Blot, Kinase Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: Thr(P)72 TPX2 antibodies are specific in Western blot for TPX2 phosphorylated at Thr72in vivo. A, specificity of Thr(P)72 TPX2 for TPX2 protein tested by siRNA. HeLa cells were transfected with control siRNA or one of two TPX2 siRNAs for 24 h and synchronized at M phase with nocodazole treatment (100 ng/ml). Cells were harvested and lysed with lysis buffer. Samples were run on SDS-PAGE, followed by Western blotting, first probed with the Thr(P)72 TPX2 Abs, then stripped and re-probed with pan-TPX2 (clone 184) Abs. Levels of actin were used as loading controls. B, bar graph quantitation for the relative expression levels of Thr(P)72 TPX2 and TPX2 in control and TPX2 siRNA-transfected cells. Each sample was compared with sample treated with control siRNA. Relative expression levels of Thr(P)72 for control siRNA, 1 ± 0; TPX2 siRNA #1 (UTR), 0.318 ± 0.085; TPX2 siRNA #2 (Cds), 0.289 ± 0.115. Relative expression levels of TPX2, control siRNA, 1 ± 0; TPX2 siRNA #1, 0.335 ± 0.0074; TPX2 siRNA #2, 0.304 ± 0.091 (mean ± S.E.). n = 4 samples, from 4 independent experiments. Unpaired Student's t test indicated all the results are significant. ***, p < 0.001. C, specificity of Thr(P)72 TPX2 tested by the use of T72A mutant and λ-PPase treatment. HeLa cells were left untransfected, transfected with an empty GFP vector, GFP-TPX2 WT, or GFP-TPX2 T72A mutant plasmids. 24 h after transfection, cells were synchronized with nocodazole for 16 h, harvested, and lysed. TPX2 immunoprecipitation was performed in each sample with TPX2 Abs (clone 183). Where indicated, IP beads were treated with λ-PPase before SDS-PAGE. The blot was first probed with the Thr(P)72 TPX2 Abs. After stripping, the same blot was re-probed with pan-TPX2 Abs (clone 184).

Article Snippet: Endogenous TPX2 was immunoprecipitated from 10 mg of total protein lysates using TPX2 Abs (clone 184, Novus Biologicals) and Protein A/G-Sepharose 4 Fast Flow beads.

Techniques: Western Blot, Transfection, Lysis, SDS Page, Quantitation Assay, Expressing, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: In vivo TPX2 phosphorylation at Thr72 is cell cycle-dependent and peaks at M phase. A, cell cycle profiles analyzed by flow cytometry analysis to confirm cell synchronization at each phase. B, after IPs with pan-TPX2 Abs (clone 184), Western blots were probed first with the Thr(P)72 TPX2 Abs and then, after stripping, re-probed with pan-TPX2 Abs (clone 184). The levels of α-actin were used as loading controls. The levels of cyclin B1 were used as positive controls to show that synchronization at M phase worked well, as indicated by the high level of cyclin B1 in M phase compared with that of S phase or non-synchronized cells. The Western blot figures are representative of 3 independent experiments. The input blot is from the mitotic samples. C, bar graphs for quantification of relative levels of Thr72 phosphorylation are shown. Non-syn, non-synchronized cells (1 ± 0); M, M phase cells (4.16 ± 0.36); S, S phase cells (1.31 ± 0.4); mean of the relative levels of Thr(P)72 TPX2/TPX2 expression ± S.D., n = 3 independent experiments; ***, non-syn versus M phase, p < 0.001; **, M versus S phase, p < 0.01; NS, not significant: non-syn versus S phase, all by unpaired Student's t test.

Article Snippet: Endogenous TPX2 was immunoprecipitated from 10 mg of total protein lysates using TPX2 Abs (clone 184, Novus Biologicals) and Protein A/G-Sepharose 4 Fast Flow beads.

Techniques: In Vivo, Flow Cytometry, Western Blot, Stripping Membranes, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: Phosphorylation of TPX2 at Thr72 is inhibited by the Cdk inhibitor roscovitine. A, the levels of Thr(P)72 were reduced by roscovitine treatment. HeLa cells were first treated with 100 ng/ml of nocodazole for 16 h and then once synchronized, treated with dimethyl sulfoxide (as a control), or 20 and 40 μm roscovitine for 30 min. Cells were harvested, lysed, and TPX2 was immunoprecipitated with pan-TPX2 Abs (clone 184). SDS-PAGE was performed and followed by Western blotting with Thr(P)72 and pan-TPX2 Abs (clone 184). B, cyclin B1 levels were also used to confirm that roscovitine-treated cells had remained in mitosis. The levels of p-Cdk/MAPK substrates (PX(S*/T*)P or (S*/T*)PX(R/K) motif) were also used to confirm the effectiveness of the treatment. Actin levels were used as loading control.

Article Snippet: Endogenous TPX2 was immunoprecipitated from 10 mg of total protein lysates using TPX2 Abs (clone 184, Novus Biologicals) and Protein A/G-Sepharose 4 Fast Flow beads.

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: Localization of Thr(P)72 TPX2 in HeLa and 293 cells. Mitotic (A and B) and interphase (C) HeLa cells were stained with Abs directed against Thr(P)72 TPX2, TPX2, and tubulin or with the Thr(P)72 Abs pre-absorbed with blocking peptide at different ratios. A, in mitotic cells, TPX2 phosphorylated at Thr72 is localized in the cytosol and does not strictly associate with the mitotic spindle. B, HeLa cells stained with pan-TPX2 and Thr(P)72 TPX2 Abs preincubated with Thr(P)72 blocking peptides. C, during interphase, Thr(P)72 TPX2 is localized in the nucleus. Note that the expression levels of Thr(P)72 are much lower in interphase cells than in mitotic cells. Note that only the Thr(P)72 signal was blocked. D, representative photographs of mitotic 293 cells transfected with GFP-TPX2 WT (WT) and GFP-TPX2 T72A (T72A). Scatter plots show the GFP signal at microtubules relative to total GFP signal. GFP-TPX2 T72A is significantly enriched on microtubules when compared with GFP-TPX2 WT (GFP-TPX2 WT (0.26 ± 0.01, n = 29) versus GFP-TPX2 T72A (0.39 ± 0.02, n = 22), group (mean ± S.E.); ***, p < 0.0001 by t test). Scale bar, 10 μm.

Article Snippet: Endogenous TPX2 was immunoprecipitated from 10 mg of total protein lysates using TPX2 Abs (clone 184, Novus Biologicals) and Protein A/G-Sepharose 4 Fast Flow beads.

Techniques: Staining, Blocking Assay, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: Effects of GFP-TPX2 T72A on the polarity of mitotic spindles in HeLa cells with or without endogenous TPX2. A, representative photographs of mitotic HeLa cells at prometaphase and metaphase with monopolar, bipolar, and multipolar mitotic spindle poles. Scale bar, 10 μm. B, Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in cells with intact levels of TPX2. C, bar graphs showing the number of cells with different mono-, bi-, or multipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. GFP-TPX2 T72A expression results in a significant increase in the percentage of cells with multipolar spindles in the presence of endogenous TPX2. ANOVA comparing the three groups shows high significance with p < 0.001. Neuman-Keuls test was used to compare each group: GFP (1.49 ± 0.47) versus T72A (12.72 ± 2.10), p < 0.001; TPX2 WT (3.36 ± 0.40) versus T72A (12.72 ± 2.10), p < 0.001; group (mean ± S.E.); ***, p < 0.001; NS, not significant (GFP versus TPX2). At least 100 cells for each set of experiments were used for quantification, 5 independent experiments were performed. Error bars indicate S.E. D, Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in HeLa cells co-transfected with GFP-vector, GFP-TPX2 WT, or GFP-TPX2 T72A together with TPX2 siRNA targeting the 3′ UTR of TPX2 mRNA. E, bar graphs showing the number of cells with different mono-, bi-, or multipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. Knockdown of TPX2 in GFP-transfected cells results in a significant 5.4% increase in multipolar spindles versus control cells without TPX2 depletion. GFP-TPX2 T72A expression produces an even greater 9.8 and 7.5% increase in the percentage of cells with multipolar spindles when compared with GFP/TPX2 siRNA and GFP-TPX2 WT/TPX siRNA, respectively. n = 3, ANOVA test was used the compare the four groups (p < 0.01). The Neuman-Keuls test was used to compare the following groups: control (with control siRNA) (2.43 ± 0.41) versus GFP (7.94 ± 1.5), p < 0.05; GFP (7.94 ± 1.5) versus TPX2 WT (10.13 ± 1.2), NS; WT (10.13 ± 1.2) versus T72A (17.67 ± 3.2), p < 0.05; GFP (7.94 ± 1.5) versus T72A (17.67 ± 3.2), p < 0.05; group (mean ± S.E.); *, p < 0.05; NS, not significant. n = at least 500 cells for each set of experiments; 3 independent experiments were performed. Error bars indicate S.E.

Article Snippet: Endogenous TPX2 was immunoprecipitated from 10 mg of total protein lysates using TPX2 Abs (clone 184, Novus Biologicals) and Protein A/G-Sepharose 4 Fast Flow beads.

Techniques: Western Blot, Staining, Expressing, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: Overactivation of Aurora A and increased spindle length, a measure of Eg5 activity, in TPX2 T72A-expressing cells. A–C show 293 mitotic cells (prometaphase/metaphase) previously transfected with GFP-TPX2 WT (WT) or GFP-TPX2 T72A (T72A) expression vectors. A, representative photographs of WT- and T72A-transfected cells stained for Thr(P)288, a phosphoresidue indicative of the activity of Aurora kinase A. Dotted circles identify the poles. Scatter plots show the P-Aurora signal at centrosomes relative to total GFP signal. GFP-TPX2 T72A induces higher Aurora A activity than GFP-TPX2 WT (GFP-TPX2 WT (0.07 ± 0.01, n = 13) versus GFP-TPX2 T72A (0.14 ± 0.03, n = 18); *, p < 0.05 by t test). B, representative photographs of the spindle length detected in mitotic 293 cells transfected with GFP-TPX2 WT or T72A. Scatter plots show the spindle length in both groups. T72A-expressing cells display longer spindles than WT-expressing cells (GFP-TPX2 WT (58.95 ± 2.12, n = 18) versus GFP-TPX2 T72A (66.67 ± 2.20, n = 21); **, p < 0.01 by t test). C, representative images of the α-tubulin signal detected in GFP-TPX2 WT and T72A-trasnfected 293 cells. No significant difference was detected between these two groups (GFP-TPX2 WT (1.93 ± 0.41, n = 15) versus GFP-TPX2 T72A (1.97 ± 0.49, n = 13); NS, non significant by t test). In all the panels: the group is the mean ± S.E.. Scale bar, 10 μm.

Article Snippet: Endogenous TPX2 was immunoprecipitated from 10 mg of total protein lysates using TPX2 Abs (clone 184, Novus Biologicals) and Protein A/G-Sepharose 4 Fast Flow beads.

Techniques: Activity Assay, Expressing, Transfection, Staining

Journal: Cancer Research

Article Title: Tumor Cell Dependence on Ran-GTP–Directed Mitosis

doi: 10.1158/0008-5472.can-07-5279

Figure Lengend Snippet: Figure 3. Aberrant mitotic spindle assembly and cell death induced by Ran targeting in tumor cells. A, siRNA silencing. HeLa cells were transfected with nontargeted (Control) or Ran-directed siRNA and analyzed by Western blotting at the indicated time intervals. B, differential modulation of Ran effector molecules. HeLa cells transfected with Ran-directed siRNA were analyzed by Western blotting at the indicated time intervals. None, nontransfected cells. *, nonspecific. C, immunofluorescence analysis. HeLa cells transfected with control (left) or Ran-directed (right) siRNA were stained for DNA (DAPI) or TPX2, and analyzed by image merging. D, time course of cell death. HeLa cells were transfected with control or Ran-directed siRNA, harvested at the indicated time intervals, and analyzed for DNA content by propidium iodide staining and flow cytometry. The percentages of cells in sub-G1, G1, or G2-M peaks are indicated.

Article Snippet: Antibodies to survivin (Novus Biologicals), Ran (Novus Biologicals, Cell Signaling, Santa Cruz Biotechnology), a-tubulin (Sigma-Aldrich), TPX2 (Novus Biologicals), RCC1 (Santa Cruz Biotechnology), cytochrome c (BD Biosciences-Clontech), Smac (ProSci), Ran-GAP1 (Abcam), X-linked inhibitor of apoptosis (XIAP; BD Biosciences), caspase-3 (Cell Signaling), or h-actin (Sigma-Aldrich) were used.

Techniques: Transfection, Control, Western Blot, Immunofluorescence, Staining, Flow Cytometry

Journal: Cell & Bioscience

Article Title: Phosphorylation of adducin-1 by TPX2 promotes interpolar microtubule homeostasis and precise chromosome segregation in mouse oocytes

doi: 10.1186/s13578-022-00943-y

Figure Lengend Snippet: Expression and subcellular localization of TPX2 during mouse oocyte meiotic maturation. A The protein content of TPX2 in oocytes at the GV, GVBD, MI, ATI, and MII stages was detected by immunoblotting. Protein loading was verified by the detection of β-actin. The molecular mass of target proteins is indicated on the right. B TPX2 was quantified for four independent repeats (normalized to β-actin, arbitrary units). Different lowercase letters above bars indicate statistical difference at p < 0.05 by ANOVA with the Tukey test. C Immunofluorescent localization of TPX2 in meiotic oocytes at various stages. D Spatial distribution of TPX2 and spindle microtubules in oocytes at different meiotic maturation stages. E Oocytes at the MI and MII stages were treated with taxol and then double-stained for TPX2 and α-tubulin. F Oocytes at the MI and MII stages were exposed to nocodazole and then co-stained for p-ADD1 and α-tubulin. Red, ADD1; green, α-tubulin; blue, DNA; Merge, overlapping of red, green, and blue. Bar, 20 μm

Article Snippet: The rabbit polyclonal anti-ADD1 antibody (Catalog# NBP1-48,611) and the mouse monoclonal anti-TPX2 antibody (Catalog# NBP2-67,265) were purchased from Novus Biologicals.

Techniques: Expressing, Western Blot, Staining

Journal: Cell & Bioscience

Article Title: Phosphorylation of adducin-1 by TPX2 promotes interpolar microtubule homeostasis and precise chromosome segregation in mouse oocytes

doi: 10.1186/s13578-022-00943-y

Figure Lengend Snippet: Effects of TPX2 depletion on oocyte maturation, spindle assembly, chromosome alignment, and the expression of p-ADD1 and ADD1. A Immunoblot probed with anti-TPX2 antibody demonstrating the depletion efficiency of TPX2-specific morpholino (MO-TPX2) in mouse oocytes. B TPX2 protein content in control-MO and TPX2-MO-injected oocytes was quantified for three independent repeats. C Representative images of oocytes cultured in vitro for 16 h after treatment with control-MO or TPX2-MO. Bar, 100 μm. D Depletion of TPX2 caused meiotic cell cycle arrest in MI in mouse oocytes. E Subcellular localization of p-ADD1, spindle morphologies, and chromosome alignment after 16 h of maturation culture in oocytes injected with control-MO or TPX2-MO. Bar, 20 μm. F The rate of the oocyte with a normal spindle, aberrant spindle, or no spindle was quantified in the control-MO and ADD1-MO-injected oocytes. G The rate of the oocyte with normal p-ADD1 localization, abnormal p-ADD1 localization, or no p-ADD1 localization was recorded in the control and ADD1 disrupted oocytes. H The depletion of TPX2 downregulated the phosphorylation of ADD1 at S726 in mouse oocytes. I The protein level of p-ADD1 in control and TPX2-depleted oocytes was quantified for three independent repeats. J Effect of TPX2 depletion on ADD1 protein expression in mouse oocytes. (K) ADD1 protein levels in control-MO and TPX2-MO-injected oocytes were quantified for three independent replicates. In B , I , K , each bar denotes the mean ± SEM of the three independent repeats, and different uppercase letters or lowercase letters above columns indicate statistical difference at p < 0.01 or p < 0.05 by student’s t -test, respectively. Whereas bars that do not share the same uppercase letter are significantly different at p < 0.01 by the chi-square test in D , F , G

Article Snippet: The rabbit polyclonal anti-ADD1 antibody (Catalog# NBP1-48,611) and the mouse monoclonal anti-TPX2 antibody (Catalog# NBP2-67,265) were purchased from Novus Biologicals.

Techniques: Expressing, Western Blot, Control, Injection, Cell Culture, In Vitro, Phospho-proteomics

Journal: Cell & Bioscience

Article Title: Phosphorylation of adducin-1 by TPX2 promotes interpolar microtubule homeostasis and precise chromosome segregation in mouse oocytes

doi: 10.1186/s13578-022-00943-y

Figure Lengend Snippet: Schematic illustration of the molecular mechanism by which ADD1 regulates spindle assembly in mouse oocytes. ADD1 is phosphorylated by TPX2 and aggregates to the spindle poles to regulate the homeostasis of interpolar microtubules to ensure the formation of functional spindles and the proper segregation of chromosomes in mouse oocytes

Article Snippet: The rabbit polyclonal anti-ADD1 antibody (Catalog# NBP1-48,611) and the mouse monoclonal anti-TPX2 antibody (Catalog# NBP2-67,265) were purchased from Novus Biologicals.

Techniques: Functional Assay