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rabbit polyclonal anti phospho cdc2 tyr15 (Cell Signaling Technology Inc)

Name: rabbit polyclonal anti phospho cdc2 tyr15
Brand: Cell Signaling Technology Inc
Rating: 96 (997 reviews)

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Cell Signaling Technology Inc rabbit polyclonal anti phospho cdc2 tyr15

(A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of <t>Cdk1</t> were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

Rabbit Polyclonal Anti Phospho Cdc2 Tyr15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase"

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

Journal: bioRxiv

doi: 10.1101/2022.01.12.476115

Figure Legend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

Techniques Used: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

(A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

Figure Legend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

Techniques Used: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

Image Search Results

(A) BJAB cells transfected with the pA3M vector or pA3M-LANA were treated with nocodazole (200 ng/mL) for 24 hours. The cells were then harvested, total cell lysate were prepared and western blot was performed using antibodies against cyclin B1, Cdc2, Tyr15-phosphorylated Cdc2 [Cdc2 (Tyr15)], and Histone H3 as a protein loading control. (B) BJAB cells transfected with the pA3M vector or pA3M-LANA were grown in 6 well culture plates and treated with nocodazole and observed for colocolaization in a confocal microscope. The result shown is the representative of 3 independent experiments. +, present; −, absent.

Journal: PLoS ONE

Article Title: Kaposi Sarcoma Herpes Virus Latency Associated Nuclear Antigen Protein Release the G2/M Cell Cycle Blocks by Modulating ATM/ATR Mediated Checkpoint Pathway

doi: 10.1371/journal.pone.0100228

Figure Lengend Snippet: (A) BJAB cells transfected with the pA3M vector or pA3M-LANA were treated with nocodazole (200 ng/mL) for 24 hours. The cells were then harvested, total cell lysate were prepared and western blot was performed using antibodies against cyclin B1, Cdc2, Tyr15-phosphorylated Cdc2 [Cdc2 (Tyr15)], and Histone H3 as a protein loading control. (B) BJAB cells transfected with the pA3M vector or pA3M-LANA were grown in 6 well culture plates and treated with nocodazole and observed for colocolaization in a confocal microscope. The result shown is the representative of 3 independent experiments. +, present; −, absent.

Article Snippet: Polyclonal rabbit anti-phospho-Tyr15 Cdc2, mouse monoclonal Cdc2, mouse monoclonal cyclin B1, anti-HA rabbit polyclonal antibody, anti-Myc mouse monoclonal antibody, rabbit polyclonal anti-β–actin, HRP conjugated mouse and rabbit polyclonal antibodies were all purchased from Santa Cruz Biotechnology (USA), IMGENEX Corporation (USA) or Cell Signaling Technology, Inc. (USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Microscopy

Nocodazole treatment reduces the level of phosphorylated Cdc2. The viral nuclear antigen LANA binds directly to Chk2, which may result in the phosphorylation of Cdc25c and sequester it in the cytoplasm. Thus, it may be unable to regulate the phosphorylation of nuclear Cdc2 resulting the activation of cyclin B-Cdc2 and progression through the G2/M phase, releasing the nocodozole induced block.

Journal: PLoS ONE

Article Title: Kaposi Sarcoma Herpes Virus Latency Associated Nuclear Antigen Protein Release the G2/M Cell Cycle Blocks by Modulating ATM/ATR Mediated Checkpoint Pathway

doi: 10.1371/journal.pone.0100228

Figure Lengend Snippet: Nocodazole treatment reduces the level of phosphorylated Cdc2. The viral nuclear antigen LANA binds directly to Chk2, which may result in the phosphorylation of Cdc25c and sequester it in the cytoplasm. Thus, it may be unable to regulate the phosphorylation of nuclear Cdc2 resulting the activation of cyclin B-Cdc2 and progression through the G2/M phase, releasing the nocodozole induced block.

Techniques: Activation Assay, Blocking Assay

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A-D) Two hours after release from a double thymidine block (DTB), synchronized HeLa S3 cells were treated with MeOH or the indicated concentrations of Pla-B. Black triangles indicate the time points of cell harvest and sample preparation (A). Cell cycle was analyzed at the indicated time points by cytometry (B). Morphology of the cells was observed under a microscope and round cells were counted at the indicated time points (C). Protein samples were prepared at the indicated time points. The protein levels of indicated proteins and phosphorylation status of Cdk1 were analysed by immunoblotting. Protein levels of α-tubulin were analysed as an internal control (D). Error bars indicate s.d. (n = 3).

Article Snippet: Mouse monoclonal anti-Cyclin A2 (#4656), rabbit polyclonal anti-Cyclin B1 (#4138), mouse monoclonal anti-cdc2 (#9116), rabbit polyclonal anti-phospho-cdc2 (Tyr15) (#9111), mouse monoclonal anti-Cyclin E1 (#4129), rabbit monoclonal anti-Plk2 (#14812), rabbit monoclonal anti-Wee1 (#13084) and rabbit monoclonal anti-p27 (#3686) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Blocking Assay, Sample Prep, Cytometry, Microscopy, Western Blot

Journal: bioRxiv

Article Title: A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase

doi: 10.1101/2022.01.12.476115

Figure Lengend Snippet: (A) Cells expressing Flag-p27* under the control of tetracycline were synchronized by a double thymidine block. The cells were treated with 1 μg/ml DOX at the same time as release from the double thymidine block. The cells were harvested at 8 h after release from the double thymidine block (G2/M phase) and then immunoprecipitation was performed using anti-DDDDK (Flag) antibodies. Flag-tagged and coimmunoprecipitated proteins were analysed by immunoblotting. (B, C) Purified Flag-tagged proteins were applied to an in vitro kinase assay reaction and kinase activities of Cyclin A2/Cdk1 (B) and Cyclin B1/Cdk1 (C) were measured. Statistical significance was assessed by the one-way ANOVA and Dunnett’s test (*: P < 0.05; **: P < 0.01; ***: P < 0.01). Error bars indicate s.d. (n = 3).

Techniques: Expressing, Blocking Assay, Immunoprecipitation, Western Blot, Purification, In Vitro, Kinase Assay

Journal: Cell Reports

Article Title: Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation

doi: 10.1016/j.celrep.2021.109565

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho-Cdk1 (Tyr15) (1:500) , Millipore , Cat# 219440; RRID: AB_564425.

Techniques: Recombinant, Plasmid Preparation, Knock-In, Clone Assay, Software

A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of Tyr15, of Cdk1 and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.

Journal: Nature Communications

Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop

doi: 10.1038/s41467-021-23657-0

Figure Lengend Snippet: A DSG (red) and the KKR (violet) motifs, and the triple K36A/K38A/R40A (KKR/AAA) Xenopus Arpp19 alanine mutant. Table depicting dephosphorylation time, B55-Arpp19 interaction and the capacity to promote mitotic entry in Arpp19-depleted extracts. B 50 ng of wild type or KKR/AAA Arpp19 mutant phosphorylated ‘in vitro’ by GwlK72M was supplemented to kinase-inactivated extracts depleted or not of the B55 protein. Arpp19 levels and of S67/S71 phosphorylation were analysed by western blotting and autoradiography, respectively. The 33 P-Arpp19/western blotting Arpp19 signal ratios were calculated using ImageJ for each time point. The percentage of the ratio remaining at each time point with respect to the ratio at 0 min was calculated and represented in bar graphs as the mean percentage ± SD; n = 3 biological independent samples. C A His-Arpp19 pulldown equivalent to 20 ng of wild type or KKR/AAA mutant was submitted to western blotting, and the amount of B55 and the levels of Arpp19 bound to the beads shown. B55/Arpp19 signal ratios were calculated using ImageJ and represented in a bar graph as the mean ratio ± SD; n = 5 biological independent samples. D Arpp19-depleted extracts were supplemented with human GwlK72M and a wild type or a KKR/AAA Arpp19 mutant and phosphorylation of Human Gwl, of Tyr15, of Cdk1 and Arpp19 ectopic levels (His-Arpp19) were assessed. E A schematic of DSG regions indicating residues mutated into alanine or threonine. Table representing results on the S67/S71 dephosphorylation time and the capacities to bind B55 or to restore mitotic entry in Arpp19-depleted egg extracts of each Arpp19 form. F Wild-type Arpp19 and the indicated mutants of the DSG motif were ‘in vitro’ phosphorylated by hGwlK72M and 1 µl sample removed at 10 and 40 min, to measure S67/S71 phosphorylation by autoradiography ( P33 Arp). The amount of Arpp19 was assessed by Coomassie blue staining (Arp) 33 . P-Arpp19 levels were normalized by Arpp19 amount using Coomassie blue signal and the increase in phosphorylation between time 10 and 40 min calculated. Data from three different experiments was then used to obtain the mean ± SD and represented in a bar graph; n = 3 biological independent samples.

Article Snippet: The antibodies used in this study are the following: Rabbit Polyclonal anti-Human Gwl , Rabbit Polyclonal Phospho-Cdc2 (Tyr15) (Cell Signaling Technology Cat#9111), Rabbit Polyclonal anti- Xenopus Arpp19 , Rabbit Polyclonal anti-PP2A/B55δ (Cell Signaling Technology Cat#2290), Rabbit Polyclonal anti- Xenopus Cdc27 , Rabbit Polyclonal anti- Xenopus Cyclin B2 , Rabbit Polyclonal anti- Xenopus Cdk1 , Rabbit Monoclonal PhosphoThr320 of PP1 (Abcam Cat#62334), Mouse Monoclonal Phospho-Erk (Cell Signaling Cat# 9106 S), Rabbit Polyclonal anti-phosphorylated Arpp19 (S67) (Cell Signaling Cat#5240 S), Rabbit Polyclonal anti-PRC1 (Santa Cruz Cat# 376982), Goat Polyclonal anti-phosphorylated PRC1 (T481) (Santa Cruz Cat#11768), Mouse Monoclonal anti-PP2A (C) subunit-α (isoform Merck Millipore Cat#05-42), Rat Polyclonal PP2A (A) subunit (Cell Signaling Cat#2260), Mouse Monoclonal anti-PP1 kindly gifted by Dr M Bollen and used in Ma et al. , Anti-PP4c (Bethyl Cat#A300-835A), Anti-PP6 (Santa Cruz Cat#393294), Goat anti-rat IgG-horseradish peroxidase (HRP) (Santa Cruz Cat#2006), HRP-conjugated anti-Rabbit secondary antibodies (Cell Signalling Technology Cat#7074), Donkey anti-goat IgG-HRP (Santa Cruz Cat# sc-2020), Rabbit Polyclonal anti-phosphorylated Arpp19 (S109/S113) (this study), and Rabbit Polyclonal anti-PP2A (B56) γ-subunit (this study).

Techniques: Mutagenesis, De-Phosphorylation Assay, In Vitro, Western Blot, Autoradiography, Staining

A B55 levels associated to 20 ng of wild type or the indicated DSG mutants of His-Arpp19-pulldowns. Arpp19 amount in these pulldowns is also shown. Data were represented as mean B55/Arpp19 ratio ± SD. Two-tailed unpaired Student’s t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for mutants Y68A, G72A and Y74A; n = 5 for the D73A, n = 6 for the wild-type form and n = 4 for the rest. B The wild type and the indicated DSG mutant forms of Arpp19 were thio-phosphorylated and used for His-pulldown. B55 and Arpp19 levels were checked by western blotting and shown. The B55/Arpp19 ratios were quantified and represented in a bar graph as mean ± SD. Two-tailed unpaired Student t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples. C The B55/Arpp19 ratios were obtained as in A for the indicated mutants and represented in a bar graph as mean ± SD. Two-tailed unpaired; p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for the wild-type form; n = 8 for the G72A mutant, n = 5 for the G72A-S71A mutant and n = 3 for the G72A-S71T mutant. D Prophase oocytes were injected or not (PG) with 50 ng of the wild-type His-Arpp19 protein or with the indicated mutant forms and, 1 h later, treated with progesterone. GVBD was then scored as a function of time. Germinal vesicle (GV) and mature oocytes (GVBD) were western blotted to determine the levels of the injected protein, as well as the phosphorylation of Gwl and of the inhibitory site of Cdk1 Tyrosine 15. E As for E , except that the indicated mutant form of Arpp19 was used.

Journal: Nature Communications

Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop

doi: 10.1038/s41467-021-23657-0

Figure Lengend Snippet: A B55 levels associated to 20 ng of wild type or the indicated DSG mutants of His-Arpp19-pulldowns. Arpp19 amount in these pulldowns is also shown. Data were represented as mean B55/Arpp19 ratio ± SD. Two-tailed unpaired Student’s t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for mutants Y68A, G72A and Y74A; n = 5 for the D73A, n = 6 for the wild-type form and n = 4 for the rest. B The wild type and the indicated DSG mutant forms of Arpp19 were thio-phosphorylated and used for His-pulldown. B55 and Arpp19 levels were checked by western blotting and shown. The B55/Arpp19 ratios were quantified and represented in a bar graph as mean ± SD. Two-tailed unpaired Student t -tests were performed in each pulldown to determine statistical relevance. p vs. wild-type Arpp19 is shown; n = 3 biological independent samples. C The B55/Arpp19 ratios were obtained as in A for the indicated mutants and represented in a bar graph as mean ± SD. Two-tailed unpaired; p vs. wild-type Arpp19 is shown; n = 3 biological independent samples for the wild-type form; n = 8 for the G72A mutant, n = 5 for the G72A-S71A mutant and n = 3 for the G72A-S71T mutant. D Prophase oocytes were injected or not (PG) with 50 ng of the wild-type His-Arpp19 protein or with the indicated mutant forms and, 1 h later, treated with progesterone. GVBD was then scored as a function of time. Germinal vesicle (GV) and mature oocytes (GVBD) were western blotted to determine the levels of the injected protein, as well as the phosphorylation of Gwl and of the inhibitory site of Cdk1 Tyrosine 15. E As for E , except that the indicated mutant form of Arpp19 was used.

Techniques: Two Tailed Test, Mutagenesis, Western Blot, Injection

A Wild type and S109/S113D Arpp19 mutant were phosphorylated ‘in vitro’ with of [γ 33 P] ATP by GwlK72M on S67/S71 and supplemented to kinase-inactivated Xenopus egg extracts. The dephosphorylation of this residue was analysed at the indicated times by autoradiography ( P33 Arp) and the amount of Arpp19 in each sample measured by western blotting (Arp). B CytoStatic Factor (CSF) egg extracts were supplemented with a trace level of Arpp19-purified protein and activated to exit meiosis by the addition of active CamKII. The levels and dephosphorylation of the indicated proteins were analysed by western blotting, whereas Cyclin B/Cdk1 activity was measured by histone H1 phosphorylation (H1K). ‘Inter’ denotes interphase egg extracts. C As for C , except that S67/S71 Arpp19 dephosphorylation and PP2A-B55 reactivation upon meiosis exit in these extracts was blocked by the concomitant addition of GwlK72M-purified protein.

Journal: Nature Communications

Article Title: The study of the determinants controlling Arpp19 phosphatase-inhibitory activity reveals an Arpp19/PP2A-B55 feedback loop

doi: 10.1038/s41467-021-23657-0

Figure Lengend Snippet: A Wild type and S109/S113D Arpp19 mutant were phosphorylated ‘in vitro’ with of [γ 33 P] ATP by GwlK72M on S67/S71 and supplemented to kinase-inactivated Xenopus egg extracts. The dephosphorylation of this residue was analysed at the indicated times by autoradiography ( P33 Arp) and the amount of Arpp19 in each sample measured by western blotting (Arp). B CytoStatic Factor (CSF) egg extracts were supplemented with a trace level of Arpp19-purified protein and activated to exit meiosis by the addition of active CamKII. The levels and dephosphorylation of the indicated proteins were analysed by western blotting, whereas Cyclin B/Cdk1 activity was measured by histone H1 phosphorylation (H1K). ‘Inter’ denotes interphase egg extracts. C As for C , except that S67/S71 Arpp19 dephosphorylation and PP2A-B55 reactivation upon meiosis exit in these extracts was blocked by the concomitant addition of GwlK72M-purified protein.

Techniques: Mutagenesis, In Vitro, De-Phosphorylation Assay, Autoradiography, Western Blot, Purification, Activity Assay

Journal: Cancer cell

Article Title: Comprehensive Molecular Characterization Identifies Distinct Genomic and Immune Hallmarks of Renal Medullary Carcinoma

doi: 10.1016/j.ccell.2020.04.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-phospho-CDK1 at tyrosine 15 , Cell Signaling Technology , Cat# 9111; RRID:AB_331460.

Techniques: Recombinant, DNA Methylation Assay, Empire Assay, Imaging, Sequencing, Plasmid Preparation, Software

The maximum noncytotoxic dose of tetrandrine suppressed the phosphorylation of ERK and MEK and activated CDC25C/CDK1/cyclinB1 pathway in xenograft tumors. (A) The expressions of key proteins of CDC25C/CDK1/cyclinB1 pathway in xenograft tumors. (B) The relative expression of key proteins of CDC25C/CDK1/cyclinB1 pathway in xenograft tumors. (C) The western blot image of p‐MEK、MEK、p‐ERK and ERK in xenograft tumors. (D) The relative expression of key proteins of p‐MEK and MEK in xenograft tumors. (E) The relative expression of key proteins of p‐ERK and ERK in xenograft tumors. (* P < .05 vs control, # P < .05 vs 6Gy, Control: control group, Tet: treatment of the maximum noncytotoxic doses of tetrandrine, 6Gy: treatment of 6Gy irradiation, 6Gy + Tet: combined treatment of 6Gy irradiation and the maximum noncytotoxic doses of tetrandrine)

Journal: Cancer Medicine

Article Title: Tetrandrine sensitizes nasopharyngeal carcinoma cells to irradiation by inducing autophagy and inhibiting MEK/ERK pathway

doi: 10.1002/cam4.3356

Figure Lengend Snippet: The maximum noncytotoxic dose of tetrandrine suppressed the phosphorylation of ERK and MEK and activated CDC25C/CDK1/cyclinB1 pathway in xenograft tumors. (A) The expressions of key proteins of CDC25C/CDK1/cyclinB1 pathway in xenograft tumors. (B) The relative expression of key proteins of CDC25C/CDK1/cyclinB1 pathway in xenograft tumors. (C) The western blot image of p‐MEK、MEK、p‐ERK and ERK in xenograft tumors. (D) The relative expression of key proteins of p‐MEK and MEK in xenograft tumors. (E) The relative expression of key proteins of p‐ERK and ERK in xenograft tumors. (* P < .05 vs control, # P < .05 vs 6Gy, Control: control group, Tet: treatment of the maximum noncytotoxic doses of tetrandrine, 6Gy: treatment of 6Gy irradiation, 6Gy + Tet: combined treatment of 6Gy irradiation and the maximum noncytotoxic doses of tetrandrine)

Article Snippet: Rabbit monoclonal anti‐cyclin B1 (ab32053, abcam), rabbit monoclonal phospho‐CDC25C‐Ser216 (E190), rabbit polyclonal phospho‐CDK1‐Tyr15 (ab47594), and mouse monoclonal CDK1 (ab18) antibodies were purchased from Abcam (USA).

Techniques: Expressing, Western Blot, Irradiation

Cdc13 HPM Cannot Localize to the SPB in Interphase (A) Top: serial dilution assays of cells with thiamine-repressible endogenous Cdc13 and either Cdc13 WT or Cdc13 HPM in the presence (endogenous Cdc13 OFF) or absence (endogenous Cdc13 ON) of thiamine. Bottom: calcofluor staining of cells expressing Cdc13 HPM and repressible endogenous Cdc13 in the presence (right) or absence (left) of thiamine is shown. Scale bars, 10 μm. (B) Representative maximum projection images of different cells of increasing size from an asynchronous population expressing exogenous Cdc13 WT/HPM -sfGFP. Endogenous Cdc13 is still expressed but is not fused to a fluorophore. Sid4-mRFP marks the SPB. Arrows show first appearance of a Cdc13-sfGFP SPB focus. The same pixel range has been applied to all images from the same channel. Scale bar, 2 μm. (C) Uninucleate cells as in (B) were analyzed for Cdc13 signal at single (interphase) or separated (mitotic) SPBs. The mean and SD of 3 replicates are shown. Uninucleate population n > 200 cells per replicate; total n = 824 total for Cdc13 WT and 954 for Cdc13 HPM . (D) Cell lengths of the entire uninucleate population and of cells with a Cdc13-sfGFP SPB focus from one replicate of (C). Error bars represent median, with whiskers delimiting the 25 th and 75 th percentiles. n = 212 total for Cdc13 WT and 214 for Cdc13 HPM . (E and F) Cell length compared to presence of Plo1-mCherry and (E) Cdc13 WT -sfGFP or (F) Cdc13 HPM -sfGFP foci at the SPB. Endogenous Cdc13 is still expressed. Data are pooled from 3 replicates; mean cell length per cohort is plotted against % of cells within that cohort with Plo1-mCherry or Cdc13-sfGFP foci at the SPB. n ≥ 89 cells per cohort. Total n = 899 cells for Cdc13 WT and 903 cells for Cdc13 HPM . (G) Cell length compared to Cdc13 HPM -sfGFP SPB foci in strains that accelerate the accumulation of Polo kinase at the SPB and in a wild-type background. Data are pooled from 3 replicates and sorted into 1-μm bins. Data are given as percentage of cells in a given bin with a Cdc13 HPM -sfGFP focus. n > 100 cells per replicate per strain. Total n = 310 cells for cut12.s11, 350 cells for cut12.T75DT78D, 244 cells for plo1.S402E, and 332 cells for wild-type (WT) cells. (H) Percentage of cells with Cdc13-sfGFP foci when released into mitosis in the presence of the temperature-sensitive plo1-24c allele. cdc2 as plo1-24c cells carrying either Cdc13 WT -sfGFP or Cdc13 HPM -sfGFP were arrested in G2 by the addition of 1 μM 1-NmPP1 for 3.5 h. Cells labeled 36°C were shifted to the plo1-24c restrictive temperature 90 min before washing out 1-NmPP1 to release into mitosis. Cells were imaged 6 min after release from the 1-NmPP1 block. The mean and SD of 3 replicates are shown. n > 75 cells per replicate per strain with a minimum of 225 cells analyzed in total. See also <xref ref-type=

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Journal: Current Biology

Article Title: The Hydrophobic Patch Directs Cyclin B to Centrosomes to Promote Global CDK Phosphorylation at Mitosis

doi: 10.1016/j.cub.2019.12.053

Figure Lengend Snippet: Cdc13 HPM Cannot Localize to the SPB in Interphase (A) Top: serial dilution assays of cells with thiamine-repressible endogenous Cdc13 and either Cdc13 WT or Cdc13 HPM in the presence (endogenous Cdc13 OFF) or absence (endogenous Cdc13 ON) of thiamine. Bottom: calcofluor staining of cells expressing Cdc13 HPM and repressible endogenous Cdc13 in the presence (right) or absence (left) of thiamine is shown. Scale bars, 10 μm. (B) Representative maximum projection images of different cells of increasing size from an asynchronous population expressing exogenous Cdc13 WT/HPM -sfGFP. Endogenous Cdc13 is still expressed but is not fused to a fluorophore. Sid4-mRFP marks the SPB. Arrows show first appearance of a Cdc13-sfGFP SPB focus. The same pixel range has been applied to all images from the same channel. Scale bar, 2 μm. (C) Uninucleate cells as in (B) were analyzed for Cdc13 signal at single (interphase) or separated (mitotic) SPBs. The mean and SD of 3 replicates are shown. Uninucleate population n > 200 cells per replicate; total n = 824 total for Cdc13 WT and 954 for Cdc13 HPM . (D) Cell lengths of the entire uninucleate population and of cells with a Cdc13-sfGFP SPB focus from one replicate of (C). Error bars represent median, with whiskers delimiting the 25 th and 75 th percentiles. n = 212 total for Cdc13 WT and 214 for Cdc13 HPM . (E and F) Cell length compared to presence of Plo1-mCherry and (E) Cdc13 WT -sfGFP or (F) Cdc13 HPM -sfGFP foci at the SPB. Endogenous Cdc13 is still expressed. Data are pooled from 3 replicates; mean cell length per cohort is plotted against % of cells within that cohort with Plo1-mCherry or Cdc13-sfGFP foci at the SPB. n ≥ 89 cells per cohort. Total n = 899 cells for Cdc13 WT and 903 cells for Cdc13 HPM . (G) Cell length compared to Cdc13 HPM -sfGFP SPB foci in strains that accelerate the accumulation of Polo kinase at the SPB and in a wild-type background. Data are pooled from 3 replicates and sorted into 1-μm bins. Data are given as percentage of cells in a given bin with a Cdc13 HPM -sfGFP focus. n > 100 cells per replicate per strain. Total n = 310 cells for cut12.s11, 350 cells for cut12.T75DT78D, 244 cells for plo1.S402E, and 332 cells for wild-type (WT) cells. (H) Percentage of cells with Cdc13-sfGFP foci when released into mitosis in the presence of the temperature-sensitive plo1-24c allele. cdc2 as plo1-24c cells carrying either Cdc13 WT -sfGFP or Cdc13 HPM -sfGFP were arrested in G2 by the addition of 1 μM 1-NmPP1 for 3.5 h. Cells labeled 36°C were shifted to the plo1-24c restrictive temperature 90 min before washing out 1-NmPP1 to release into mitosis. Cells were imaged 6 min after release from the 1-NmPP1 block. The mean and SD of 3 replicates are shown. n > 75 cells per replicate per strain with a minimum of 225 cells analyzed in total. See also Figure S2 and .

Article Snippet: Rabbit polyclonal anti-Cdc2-Y15P , Cell Signaling Technologies , Cat# 9111; RRID: AB_331460.

Techniques: Serial Dilution, Staining, Expressing, Labeling, Blocking Assay

Journal: Current Biology

Article Title: The Hydrophobic Patch Directs Cyclin B to Centrosomes to Promote Global CDK Phosphorylation at Mitosis

doi: 10.1016/j.cub.2019.12.053

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Cdc2-Y15P , Cell Signaling Technologies , Cat# 9111; RRID: AB_331460.

Techniques: Recombinant, Protease Inhibitor, Mass Spectrometry, Plasmid Preparation, Software

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1) Product Images from "A truncated form of the p27 CDK inhibitor translated from pre-mRNA causes cell cycle arrest at G2 phase"

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