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).

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

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

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.

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: Two Tailed Test, Mutagenesis, Western Blot, Injection

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.

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, 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

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

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Scheme of the experimental protocol. b TTC-stained rostral and caudal brain sections from vehicle-treated Ctrl and Cdk1-cKO mice and R-roscovitine-treated Ctrl mice after 1-h MCAO and 24 h of reperfusion. Scale bar = 1 mm. c Rostral and caudal quantification of infarct size (% brain area) (data represent means ± SEM; n = 6-10; *p<0.05; **p<0.005; *** p < 0.001; ANOVA with Bonferroni’s post hoc). d Quantification of neuroscore scale (data represents means ± SEM; n = 6–10; * p < 0.01; ANOVA with Bonferroni’s post hoc)

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Staining

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Scheme of the experimental protocol. b Representative confocal microscopy images and quantitative results showing no differences in the number of CC3-positive cells between normoxic and OGD condition 4 h following OGD, scale bar = 30 µm and field size = 120 µm × 70 µm (data represent means ± SEM; n = 4; ns; Student’s t -test). c Representative confocal microscopy images and quantitative results illustrating significant differences in the number of CC3-positive cells (data represent means ± SEM; n = 4; *** p < 0.001; Student’s t -test) and survival (MTT assay) (data represent means ± SEM; n = 5; * p < 0.01; Student’s t -test) between normoxic and OGD condition at 24 h following OGD. d Representative confocal images showing OGD-induced Cdk1 expression 4 h following OGD compared with normoxic condition, scale bar = 30 µm. Quantitative results represent the percentage of living (CC3-negative) neurons expressing Cdk1, field size = 120 µm × 70 µm (data represent means ± SEM; n = 5; *** p < 0.001; Student’s t -test). Western blot analysis demonstrated increased Cdk1 expression 4 h following OGD

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Confocal Microscopy, MTT Assay, Expressing, Western Blot

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Representative confocal images illustrating OGD-induced Cdk1 neuronal expression, scale bar = 30 µm. Quantitative results represent the percentage of Tuj1-positive living neurons (green) expressing Cdk1 (red), field size = 140 µm × 90 µm (data represent means ± SEM; n = 4; *** p < 0.001; ANOVA with Bonferroni’s post hoc). Western blot analysis indicated increased Cdk1 expression 1, 2 and 4 h following OGD. b Representative confocal images illustrating the number of Tuj1-positive (green) and CC3-positive (red) cells in the different conditions/genotypes 24 h following OGD, scale bar = 30 µm. Quantitative results for neuronal survival represents the number of Tuj1-positive/CC3-negative cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 5; *** p < 0.001; one-way ANOVA with Bonferroni’s post hoc). MTT assay confirm significant difference regarding neuronal survival between the different conditions 24 h following OGD (data represent means ± SEM; n = 5; *p<0.05; **p<0.01 ; one-way ANOVA with Bonferroni’s post hoc). Quantitative results for neuronal death represent the number of CC3-positive cells, field size = 230 µm × 150 µm (data represent means ± SEM; n = 4; *p<0.05; ** p < 0.005; one-way ANOVA). Western blot analysis showed decreased C-Parp (89 kDa) protein expression in Cdk1-cKO OGD-treated cultures as compared with Ctrl OGD-treated cultures

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Expressing, Western Blot, MTT Assay

Journal: Cell Death Discovery

Article Title: Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death

doi: 10.1038/s41420-018-0044-7

Figure Lengend Snippet: a Photography of 2 mm thick TTC-stained mouse brain coronal section 24 h after a transient 1 h MCAO, showing the healthy cortex (red), ischemic core (white) and peri-infarct area (between red and white) in the ipsilateral cortex. b Immunohistochemistry images showing NeuN staining in the healthy part of the ipsilateral cortex and the absence of NeuN staining in the ischemic core. In between, the peri-infarct area shows re-expression of Cdk1 and P-Cdk1. Scale bar = 100 µm

Article Snippet: The following antibodies were used at the indicated dilutions: rabbit monoclonal anti-β-III tubulin (Tuj1) (1:500; BioLegend cat# 801202, RRID:AB_10063408), rabbit polyclonal anti-phospho-Cdk1 (Thr161) (1:1000; Cell Signaling Technology cat# 9111S, RRID:AB_331460), rabbit anti-Cdk1 (1:500; Atlas Antibodies cat# HPA003387, RRID:AB_1846356), rabbit anti-CC3 (1:1000; Cell Signaling Technology cat# 9661, RRID:AB_2341188), mouse anti-GFAP (1:500; Sigma-Aldrich cat# G3893, RRID:AB_477010), mouse monoclonal anti-Cdk1 (1:500; BD Biosciences cat# 612306, RRID:AB_399621), mouse anti-NeuN (1:500, Millipore cat# MAB377, RRID:AB_2298772).

Techniques: Staining, Immunohistochemistry, Expressing