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: 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

Journal: Current biology : CB

Article Title: Nek9 Phosphorylation Defines a New Role for TPX2 in Eg5-Dependent Centrosome Separation before Nuclear Envelope Breakdown.

doi: 10.1016/j.cub.2017.11.046

Figure Lengend Snippet: Figure 1. TPX2 Is Necessary for Eg5 Prophase Centrosome Localization and Centrosome Separation and Interacts with Eg5 in a Eg5 [Ser1033]-, Nek9-, and Phospho-Dependent Manner (A) HeLa cells were transfected with the indicated small interfering RNAs (siRNAs). Cells were stained for Eg5 plus pericentrin as a centrosome marker or tubulin, while DAPI was used to visualize DNA. TPX2 downregulation was confirmed by western blot. Representative cells and quantifications of Eg5 and tubulin at centrosomes and of centrosome separation in prophase are shown. Scale bar, 10 mm. (B) HeLa cells were transfected with either GFP or the indicated GFP-TPX2 constructs. GFP was immunoprecipitated from cell lysates, and immunoprecipitates were analyzed by western blotting. (C) HeLa cells were synchronized and transfected with either GFP or GFP-TPX2 and the indicated FLAG-Eg5 constructs. GFP was immunoprecipitated from mitotic cell lysates, and immunoprecipitates were analyzed by western blotting. (D) HeLa cells were synchronized and transfected with either GFP (–) or GFP-TPX2 (TPX2). GFP immunoprecipitates from mitotic cell lysates were left untreated or incubated with lambda phosphatase buffer with or without phosphatase for 15 min at 30C. After washing, proteins in the immunoprecipitates were analyzed by western blot. (E) HeLa cells were infected with lentivirus encoding the indicated shRNAs and, after selection, transfected with either GFP or GFP-TPX2. GFP was immuno- precipitated from cell lysates, and the precipitated proteins were analyzed by western blotting. Nek9 downregulation was confirmed by western blot. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Nek9 [20] N/A Rabbit anti-TPX2 [32] N/A Mouse anti-TPX2 [43] N/A Rabbit anti-XTPX2 [44] N/A Rabbit anti-mouse TPX2 [45] N/A Mouse anti-GADPH Santa Cruz Cat# sc-47724; RRID: AB_627678 Rabbit anti-RHAMM Santa Cruz Cat# sc-25488; RRID: AB_2118510 Mouse anti-XTPX2 Abyntek Biopharma N/A Rabbit anti-GFP Torrey Pines Cat# TP401; RRID: AB_10013661 Mouse anti-GFP Thermo Fisher Cat# A11120; RRID: AB_221568 Mouse anti-FLAG Sigma Cat# F3165; RRID: AB_259529 Mouse anti-a-tubulin Sigma Cat# T6199; RRID: AB_477583 Mouse anti- b-tubulin Sigma Cat# T4026; RRID: AB_477577 Mouse anti-g-tubulin Sigma Cat# T6557; RRID: AB_477584 Rabbit anti-pericentrin Abcam Cat# ab4448; RRID: AB_304461 Rabbit anti-lamin B1 Abcam Cat# ab16048; RRID: AB_443298 Rabbit anti-importin-b Abcam Cat# ab36775; RRID: AB_881759 Mouse anti-Eg5 BD Bioscience Cat# 611186; RRID: AB_398720 Rabbit anti-Eg5 Genetex Cat# GTX30692; RRID: AB_625879 Goat anti-rabbit IgG - HRP R&D Cat# HAF008; RRID: AB_357235 Goat anti-mouse IgG - HRP R&D Cat# HAF007; RRID: AB_357234 Goat anti-rabbit IgG - Alexa Fluor 488 Invitrogen Cat# A11008; RRID: AB_143165 Goat anti-mouse IgG - Alexa Fluor 488 Invitrogen Cat# A11029; RRID: AB_138404 Goat anti-rabbit IgG - Alexa Fluor 555 Invitrogen Cat# A21429; RRID: AB_141761 Goat anti-mouse IgG - Alexa Fluor 555 Invitrogen Cat# A21422; RRID: AB_141822 Goat anti-rabbit IgG - Alexa Fluor 647 Invitrogen Cat# A21244; RRID: AB_141663 Bacterial and Virus Strains E. coli BL21pRep4 [32] N/A E. coli BL21(DE3)pLysS Promega Cat# L1195 Chemicals, Peptides, and Recombinant Proteins Nocodazole Sigma Cat# M1404 Monastrol Sigma Cat# M8515 STLC Sigma Cat# 164739 Thymidine Sigma Cat# T1895 Puromycin Sigma Cat# P8833 Triton X-100 Sigma Cat# X100 NP40 (IGEPAL CA-630) Sigma Cat # I8896 DAPI Sigma Cat# D9542 FLAG peptide Sigma Cat# F3290 Hoechst 33342 Thermo Fisher Cat# H3570 Lipofectamine 2000 Thermo Fisher Cat# 11668027 Lipofectamine RNAiMax Thermo Fisher Cat# 13778-075 Polyethyleneimide Polyscience Cat# 23966 Lambda phosphatase New England Biolabs Cat# P0753S (Continued on next page) e1 Current Biology 28, 1–9.e1–e4, January 8, 2018

Techniques: Transfection, Staining, Marker, Western Blot, Construct, Immunoprecipitation, Incubation, Infection, Selection

Journal: Current biology : CB

Article Title: Nek9 Phosphorylation Defines a New Role for TPX2 in Eg5-Dependent Centrosome Separation before Nuclear Envelope Breakdown.

doi: 10.1016/j.cub.2017.11.046

Figure Lengend Snippet: Figure 4. Nek9 Sites Control TPX2 Nucleocytoplasmic Distribution and Prophase Centrosome Localization and Are Necessary for Eg5 Accu- mulation and Centrosome Separation in Prophase (A) HeLa cells were synchronized and transfected with the indicated GFP-TPX2 constructs. Mitotic cells were fixed and stained with anti-GFP and anti-pericentrin plus DAPI to visualize the centrosomes and DNA, respectively. GFP intensity at centrosomes was quantified. Representative cells are shown. Scale bar, 10 mm. (B) HeLa cells were synchronized and transfected with the indicated siRNAs and plasmids expressing GFP alone (–) or fused to the indicated TPX2 forms. Cells were fixed and stained for Eg5, GFP, pericentrin, and DNA with DAPI and imaged in a confocal microscope. Cell-cycle phase of GFP-positive cells was assessed by DNA morphology. Representative cells and quantifications of Eg5 intensity and intercentrosomal distances for prophase GFP-positive cells are shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Nek9 [20] N/A Rabbit anti-TPX2 [32] N/A Mouse anti-TPX2 [43] N/A Rabbit anti-XTPX2 [44] N/A Rabbit anti-mouse TPX2 [45] N/A Mouse anti-GADPH Santa Cruz Cat# sc-47724; RRID: AB_627678 Rabbit anti-RHAMM Santa Cruz Cat# sc-25488; RRID: AB_2118510 Mouse anti-XTPX2 Abyntek Biopharma N/A Rabbit anti-GFP Torrey Pines Cat# TP401; RRID: AB_10013661 Mouse anti-GFP Thermo Fisher Cat# A11120; RRID: AB_221568 Mouse anti-FLAG Sigma Cat# F3165; RRID: AB_259529 Mouse anti-a-tubulin Sigma Cat# T6199; RRID: AB_477583 Mouse anti- b-tubulin Sigma Cat# T4026; RRID: AB_477577 Mouse anti-g-tubulin Sigma Cat# T6557; RRID: AB_477584 Rabbit anti-pericentrin Abcam Cat# ab4448; RRID: AB_304461 Rabbit anti-lamin B1 Abcam Cat# ab16048; RRID: AB_443298 Rabbit anti-importin-b Abcam Cat# ab36775; RRID: AB_881759 Mouse anti-Eg5 BD Bioscience Cat# 611186; RRID: AB_398720 Rabbit anti-Eg5 Genetex Cat# GTX30692; RRID: AB_625879 Goat anti-rabbit IgG - HRP R&D Cat# HAF008; RRID: AB_357235 Goat anti-mouse IgG - HRP R&D Cat# HAF007; RRID: AB_357234 Goat anti-rabbit IgG - Alexa Fluor 488 Invitrogen Cat# A11008; RRID: AB_143165 Goat anti-mouse IgG - Alexa Fluor 488 Invitrogen Cat# A11029; RRID: AB_138404 Goat anti-rabbit IgG - Alexa Fluor 555 Invitrogen Cat# A21429; RRID: AB_141761 Goat anti-mouse IgG - Alexa Fluor 555 Invitrogen Cat# A21422; RRID: AB_141822 Goat anti-rabbit IgG - Alexa Fluor 647 Invitrogen Cat# A21244; RRID: AB_141663 Bacterial and Virus Strains E. coli BL21pRep4 [32] N/A E. coli BL21(DE3)pLysS Promega Cat# L1195 Chemicals, Peptides, and Recombinant Proteins Nocodazole Sigma Cat# M1404 Monastrol Sigma Cat# M8515 STLC Sigma Cat# 164739 Thymidine Sigma Cat# T1895 Puromycin Sigma Cat# P8833 Triton X-100 Sigma Cat# X100 NP40 (IGEPAL CA-630) Sigma Cat # I8896 DAPI Sigma Cat# D9542 FLAG peptide Sigma Cat# F3290 Hoechst 33342 Thermo Fisher Cat# H3570 Lipofectamine 2000 Thermo Fisher Cat# 11668027 Lipofectamine RNAiMax Thermo Fisher Cat# 13778-075 Polyethyleneimide Polyscience Cat# 23966 Lambda phosphatase New England Biolabs Cat# P0753S (Continued on next page) e1 Current Biology 28, 1–9.e1–e4, January 8, 2018

Techniques: Control, Transfection, Construct, Staining, Expressing, Microscopy