Journal: PLoS ONE

Article Title: c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

doi: 10.1371/journal.pone.0025467

Figure Lengend Snippet: ( A ) Pt1- (dashed line) and control D11-LCL tet cells (solid line) grown in the absence of Tc for 5 days were plated at 1×10 6 cells/mL in Tc media and cultured in parallel for 6 days. Cells were withdrawn at days 1, 3 and 6 cells and counted using Trypan-Blue exclusion to determine the total number of viable cells. ( B ) 1×10 6 Pt1- and control D11- and C2-LCL tet cells were stained with CFSE and cultured in Tc media (pink line), absence of Tc (for 5 days prior to CFSE staining) (grey peak) or absence of Tc plus sCD40L (500 ng/mL) (green line) for 6 days. At day 4 (upper panel) and day 6 (lower panel) cells were analyzed for proliferation by flow cytometry. ( C ) Pt1- and control D11-LCL tet cells (2×10 5 ) grown in Tc media were analyzed for the expression of phosphorylated-CDC2 by intra-cellular staining. Numbers shown are the mean fluorescence intensity (MFI) of cells expressing high levels of phosphorylated CDC2. Graph is representative of three independent experiments.

Article Snippet: Anti-S6 mAb, rabbit polyclonal anti-caspase 4 and rabbit polyclonal anti-phospho-CDC2 were purchased from Cell Signaling.

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing, Fluorescence

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: The following primary antibodies were used: mouse monoclonal anti-SMARCB1 antibody clone 2C2 (Sigma-Aldrich; SAB4200202), rabbit polyclonal anti-phospho-Histone H2A.X at serine 139 (γH2AX; Cell Signaling Technology; 2577), mouse monoclonal anti-c-MYC antibody 9E10 (Santa Cruz Biotechology; sc-40), rabbit polyclonal anti-PARP (Cell Signaling Technology; 9542), goat polyclonal anti-ATR (Santa Cruz Biotechnology; sc-1887), rabbit polyclonal anti-phospho-ATR at serine 428 (Cell Signaling Technology; 2853), mouse monoclonal anti-TP53 (Santa Cruz Biotechnology; sc-126), rabbit polyclonal anti-phospho-TP53 at serine 15 (Cell Signaling Technology; 9284), mouse monoclonal anti-actin (Santa Cruz Biotechnology; sc- 47778), rabbit polyclonal anti-phospho-CDK1 at tyrosine 15 (Cell Signaling Technology; 9111), mouse monoclonal anti-RPA 32 kDa subunit 9H8 (Santa Cruz Biotechology; sc-56770), rabbit polyclonal anti-phospho-RPA32 at serines 4 and 8 (Bethyl Laboratories; A300–245A), mouse monoclonal anti-FANCD2 (Santa Cruz Biotechology; sc-20022).

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

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: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: RNAi duplex sequences

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: ISL1 interacts with CDK1. a–c Co-IP assays were performed to measure the ISL1/CDK1/cyclinB1 complex in MGC803 cells. Lysate was immunoprecipitated with antibodies against ISL1, CDK1, or cyclinB1 and analyzed by immunoblotting. IgG: immunoglobulin G. d Full-length or truncated ISL1 was used to construct GST-fusion proteins (Top) for pull-down assays with CDK1 protein (Bottom)

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Construct

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: ISL1 is serine-phosphorylated. a MGC803 cells lysates were immunoprecipitated (IP) using anti‑ISL1 and analyzed by immunoblotting using antibody against phosphorylated serine and ISL1 (HC: heavy chain, LC: light chain). b Alignments of the cluster of ISL1; S269 fit the CDK1 phosphorylation consensus, and (S/T)PX(R/K) sequences (red shade) are shown

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: CDK1 mediates ISL1 phosphorylation at serine 269. a MGC803 cells transfected with plasmid encoding CDK1 or control vector (left), negative control or CDK1 si-RNA (right). Lysate was analyzed by immunoblotting with antibodies against the indicated proteins. b MGC803 cells were transfected with plasmids encoding dominant‑negative mutant CDK1 (DN‑CDK1) or CDK1, and lysate was analyzed by immunoblotting with antibodies against the indicated proteins. c MKN28 cells expressing wild-type ISL1 or ISL1-S269A were transfected with plasmid encoding CDK1 or control vector. Cell lysates were immunoblotted with antibodies against the indicated proteins. d MKN28 cells were transfected with plasmids encoding wild-type ISL1 or ISL1-S269A and treated with CDK1 inhibitor RO-3306 or DMSO. Lysates were analyzed by immunoblotting with antibodies against the indicated proteins. GAPDH served as the internal control. e Representative IHC staining of ISL1-S269-p expression in GC (right, n = 60) and paired adjacent tissues (left, n = 60). f Relationship between expression of ISL1-S629-p and overall survival in gastric cancer

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Plasmid Preparation, Negative Control, Western Blot, Dominant Negative Mutation, Expressing, Immunohistochemistry

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: Phosphorylated ISL1 showed improved localization on cyclinB1 and cyclinB2 promoters. a ChIP assay analysis and PCR gel electrophoresis analysis of ISL-1 recruitment to the cyclinB1 and cyclinB2 promoters in MGC803 cells. b ChIP-re-IP assay was performed with anti-ISL-1 or rabbit IgG antibodies and then with anti-CDK1 or IgG antibodies for immunoprecipitation using chromatin harvested from MGC803 cells. c , d Quantitative chromatin immunoprecipitation analysis of cyclinB1 and cyclinB2 promoters in MKN28 cell line transfected with control vector or expressing wild-type ISL1 or ISL1-S269A plasmids. Data are expressed as mean ± s. d. from three individual experiments. (* p < 0.05, WT vs. control, # p < 0.05, S269A vs. control, $ p < 0.05, WT vs. S269A)

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Nucleic Acid Electrophoresis, Immunoprecipitation, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Expressing

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: Phosphorylation Ser 269 of ISL1 increases transactivation potency. a , b Effect of CDK1 activity on the transcriptional activity of wild-type ISL1 or S269A ISL1. Transcriptional activity of wild-type ISL1 and S269A ISL1 on cyclinB1-luc and cyclinB2-luc determined by luciferase reporter assay in MKN28 cells. c Cell proliferation was studied by CCK-8 analysis of MKN28 cells transfected with plasmids encoding control vector and wild-type ISL1 or ISL1-S269A. d Cell cycle distributions were analyzed by flow cytometry in MKN28 transfected with plasmids encoding control vector and wild-type ISL1 or ISL1-S269A. The data represent 3 independent experiments, each performed in triplicate. Each bar represents mean ± SD. (* p < 0.05, WT vs. control, # p < 0.05, S269A vs. control, $ p < 0.05, WT vs. S269A)

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Luciferase, Reporter Assay, CCK-8 Assay, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Molecular Medicine

Article Title: Phosphorylation of islet-1 serine 269 by CDK1 increases its transcriptional activity and promotes cell proliferation in gastric cancer

doi: 10.1186/s10020-021-00302-6

Figure Lengend Snippet: Phosphorylation of ISL1 increases self-stability but does not increase its interaction with CDK1. a NIH3T3 cells were transfected with wild-type ISL1 or ISL1-S269A plasmids. After 48 h, cells were treated with CHX (100 µg/ml) for 12 h. Whole cell extracts were harvested for western blotting. b Co-IP assays were performed in NIH3T3 cells to detect the interaction between ISL1 (wild-type or mutants) and CDK1. GAPDH served as a loading and negative control. c Schematic representation of positive feedback regulation by the ISL1-CDK1/cyclin B1/2 complex in GC. Full arrow: confirmed; broken arrow: to be verified

Article Snippet: Monoclonal anti-CDK1 (#9116), GAPDH (#2118), rabbit polyclonal anti-CDK1-T161P (#9114), and mouse monoclonal anti-cyclin B1 (#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay, Negative Control