Journal: Protein Expression and Purification

Article Title: Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli

doi: 10.1016/j.pep.2014.11.006

Figure Lengend Snippet: Comparison of specific activity of human CES1 toward pNPA.

Article Snippet: Insect cells, Sf9 and Sf21, are also preferred for expressing CES1, which is accomplished using a baculovirus expression system .

Techniques: Activity Assay

Journal: eLife

Article Title: Binding and sequestration of poison frog alkaloids by a plasma globulin

doi: 10.7554/eLife.85096

Figure Lengend Snippet:

Article Snippet: Cell line ( Spodoptera frugiperda ) , Sf9 insect cell culture , Kemp Proteins , , Proprietary insect cell expression technology.

Techniques: Sequencing, Cell Culture, Expressing, Clinical Proteomics, Generated, Recombinant, Plasmid Preparation, Cloning, Purification, Mutagenesis, Labeling, RNA Sequencing, Produced, Software, Mass Spectrometry, Molecular Weight, Microscale Thermophoresis, Immunohistochemistry

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for β-catenin and IKKβ proteins in control or β-catenin–deficient C3H/10T1/2 cells infected with control or WT IKKβ virus. (B and C) Oil Red O staining (B) and qPCR analysis (C) of control and β-catenin–deficient C3H/10T1/2 cells induced by an adipogenic cocktails (n = 3). Scale bar: 100 μm. (D and E) ALP staining (D) and qPCR analysis (E) of control and β-catenin–deficient C3H/10T1/2 cells induced by an osteogenic cocktails (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by 2-way ANOVA (C and E). **P < 0.01, ***P < 0.001.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Western Blot, Infection, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Control or IKKβ-deficient C3H/10T1/2 cells were treated with vehicle or 100 nM PS-341. β-Catenin proteins were immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. (B and C) Immunoblotting for nuclear β-catenin proteins (B) and β-catenin reporter activity (C) in control or IKKβ-deficient C3H/10T1/2 cells. (D and E) Immunoblotting for ubiquitinated β-catenin proteins (D) and nuclear β-catenin proteins (E) in control or BMS-345541–treated C3H/10T1/2 cells. (F and G) Immunoblotting for ubiquitinated β-catenin proteins (F) and nuclear β-catenin proteins (G) in C3H/10T1/2 cells infected with control, IKKβ WT, or IKK KM virus. (H and I) Immunoblotting for ubiquitinated β-catenin levels (H) and nuclear β-catenin proteins (I) in control or FFA-treated C3H/10T1/2 cells. (J) Immunoblotting for ubiquitinated β-catenin proteins in control or IKKβ-deficient C3H/10T1/2 cells treated with vehicle or FFAs. Error bars represent ± SEM. Significance was determined by Student’s t test (C). ***P < 0.001.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Infection

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) The sequence of a conserved 6–amino acid motif found in β-catenin and IκB family members. (B) Immunoblotting for FLAG-tagged IKKβ and HA-tagged β-catenin proteins after immunoprecipitation using control IgG or antibodies against FLAG or HA proteins in C3H10T1/2 cells and HEK 293T cells. (C) In vitro phosphorylation of purified GST–β-catenin proteins by IKKβ in the presence of γ-[32P]ATP. (D–F) GST–β-catenin proteins phosphorylated by IKKβ in vitro were tryptic digested and analyzed by mass spectrometry. Figures show the recovered phosphorylated 30-residue fragment of β-catenin (residues 20–49). Tandem mass spectrum of the recovered peptide phosphorylated at ser45 residue (D). The peptides phosphorylated at ser33 and ser37 residues were coeluted. Color codes are used to mark fragment ions that allows distinguishing phosphorylation of ser33 (blue) and ser37 (red) (E). Tandem MS ion traces (10 ppm extraction) for the nonphosphorylated peptide, ser45- and ser33/ser37-phosphorylated peptides, and AUCs shown in italics (F).

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Sequencing, Western Blot, Immunoprecipitation, In Vitro, Purification, Mass Spectrometry

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) GST–β-catenin and indicated mutant proteins were phosphorylated by IKKβ in vitro and analyzed by immunoblotting using anti–phospho-ser33, -ser37 or -ser45 β-catenin antibodies. (B) Immunoblotting for phosphorylated β-catenin proteins in C3H/10T1/2 cells infected with control, WT IKKβ, and IKKβ KM virus. (C) Immunoblotting for phosphorylated β-catenin proteins in C3H/10T1/2 cells treated with vehicle or FFAs. (D) Immunoblotting for phosphorylated β-catenin proteins in control or IKKβ-deficient C3H/10T1/2 cells treated with vehicle or LPS. (E) GST–β-catenin and indicated mutant proteins were phosphorylated by IKKβ in vitro. The reaction substrates were subjected for cell-free ubiquitination assays and analyzed by immunoblotting.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Mutagenesis, In Vitro, Western Blot, Infection

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for IKKβ proteins in BMMSCs of IKKβF/F and Prrx1Cre+IKKβF/F mice. (B–G) BMMSCs were isolated from IKKF/F and Prrx1Cre+IKKβF/F mice. Oil Red O staining (B) and qPCR analysis (C) of BMMSCs induced by an adipogenic cocktail (n = 3). ALP staining (D) and qPCR analysis (E) of BMMSCs induced by an osteogenic cocktail (n = 3). Immunoblotting for ubiquitinated β-catenin (F) and nuclear β-catenin proteins (G) of isolated BMMSCs. Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (C and E). *P < 0.05; **P < 0.01.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Western Blot, Isolation, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A–C) Adipose stem cells were isolated from s.c. adipose tissue of heathy human subjects. Immunoblotting for IKKβ and phosphorylated β-catenin proteins (A), ubiquitinated β-catenin proteins (B), and nuclear β-catenin proteins (C) in human adipose stem cells infected with control, WT IKKβ, and IKKβ KM virus. (D and E) Oil Red O staining (D) and qPCR analysis (E) of human adipose stem cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (F and G) Alizarin Red S staining (F) and qPCR analysis (G) of human adipose stem cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. (H and I) Immunoblotting for phosphorylated β-catenin proteins (H) and ubiquitinated β-catenin proteins (I) in human adipose stem cells treated with vehicle control or FFAs. (J and K) Oil Red O staining (J) and qPCR analysis (K) of control or FFA-treated human adipose stem cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (L and M) Alizarin Red S staining (L) and qPCR analysis (M) of control or FFA-treated human adipose stem cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (K and M) or 1-way ANOVA (E and G). *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Isolation, Western Blot, Infection, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) s.c. adipose tissues were isolated from a cohort of nondiabetic human subjects. Correlation between adipose IKKβ mRNA levels and BMI (n = 27). The correlation was analyzed by Pearson correlation coefficient. (B) IKKβ mRNA levels in adipose tissue of nonobese and obese human subjects (n = 12–15). (C and D) Immunoblotting (C) and densitometric quantification (D) of proteins in adipose tissue of nonobese and obese human subjects (n = 7). Error bars represent ± SEM. Significance was determined by Student’s t test (B and D). *P < 0.05; **P < 0.01, ***P < 0.001. (E) Schematic representation of the mechanism through which IKKβ reciprocally regulates adipogenesis and osteogenesis in MSCs. Activation of IKKβ by stimuli such as FFAs or inflammation cytokines phosphorylates serine-33, -37, and -45 of β-catenin to prime it for β-TrCP–mediated ubiquitination and degradation, leading to increased adipogenic differentiation and reduced osteogenic differentiation of MSCs.

Article Snippet: After in vitro phosphorylation by IKKβ protein (SignalChem) in kinase buffer containing 100 nM of ATP, the human β-catenin proteins were separated by SDS-PAGE and excised followed by trypsinization as described previously ( 62 ).

Techniques: Isolation, Western Blot, Activation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: VEGF-induced vascular permeability is reduced upon CRISPR/Cas9-mediated knockout of Stat3 in zebrafish. (A) VEGF-inducible zebrafish were crossed to Stat3 +/− (heterozygous) zebrafish to generate VEGF-inducible; Stat3 +/− double transgenic fish, which were intercrossed to generate VEGF-inducible; Stat3 −/− (KO) zebrafish. (B) CRISPR/Cas9-generated Stat3 KO zebrafish (bottom) display no overt vascular defects relative to wild-type (WT) zebrafish (top). The vascular system of 3 days post-fertilization (dpf) zebrafish was visualized by microangiography with 2000 kDa FITC-dextran. Representative images of at least three zebrafish per group are shown. Scale bars: 100 μm. (C) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf Stat3 +/+ (negative controls without VEGF induction; left) , VEGF-induced, Stat3 +/+ (middle) and VEGF-induced, Stat3 −/− (right) zebrafish. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at room temperature (RT). Scale bars: 50 μm. (D) Quantitative analysis of vascular permeability upon VEGF stimulation in WT Stat3 +/+ ( n =30) and KO Stat3 −/− ( n =9) zebrafish. Mean±s.e.m., unpaired, two-tailed Student's t -test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, CRISPR, Knock-Out, Transgenic Assay, Generated, Marker, Microscopy, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Endothelial cell-specific STAT3 knockout mice exhibit decreased VEGF-induced permeability. (A) Images of footpads from WT and endothelial cell-specific STAT3 knockout (STAT3 ECKO ) mice following tail vein injection with 1% Evans Blue dye and human recombinant VEGF-165 protein (2.5 µg/ml; left footpads) or PBS vehicle (right footpads) being injected into the root of the footpad. (B,C) Quantitation of Evans Blue leakage in Tie2-Cre negative; STAT3 flox/flox (WT) and Tie2-Cre positive; STAT3 flox/flox (STAT3 ECKO ) mice. n =7 mice in WT group and n =6 mice in STAT3 ECKO group. Each mouse was injected with PBS on the right anterior and posterior footpads and VEGF on the left anterior and posterior footpads. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. A.U., arbitrary units.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Knock-Out, Permeability, Injection, Recombinant, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Pharmacological inhibition of STAT3 stabilizes endothelial barrier integrity following VEGF stimulation in human endothelial cells. (A) Serum-starved human umbilical vein endothelial cells (HUVECs) were pretreated with DMSO (vehicle control) for 1 h, 30 µM AQ for 4 h, or 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 2 or 5 min. Lysates were immunoblotted. Densitometry was performed, and the values below the rows of bands represent the ratio of phosphorylated protein to respective total protein. (B) Human VEGF-165 recombinant protein (VEGF; 25 ng/ml) stimulation of HUVECs promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows; left column; DMSO vehicle control pretreatment for 1 h prior to VEGF stimulation). ZO-1 organization is maintained upon pretreatment with 30 μM AQ for 4 h (magenta arrows; middle column) or 10 μM PYR for 1 h (magenta arrows; right column) prior to VEGF stimulation. Nuclei were stained with DAPI (blue). (C) Serum-starved human pulmonary artery endothelial cells (HPAECs) were pretreated with 10 µM PYR for 1 h prior to VEGF (25 ng/ml) stimulation for 0, 5 or 30 min. VEGF stimulation promotes disorganization of ZO-1 (green) at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained when HPAECs were pretreated with PYR (magenta arrows). Nuclei were stained with DAPI (blue). (D) VEGF (25 ng/ml) stimulation of human lung microvascular endothelial cells (HMVEC-Ls) promotes ZO-1 (green) disorganization at endothelial cell junctions (yellow arrows). ZO-1 organization is maintained upon pretreatment with 20 μM PYR for 6 h prior to VEGF stimulation (magenta arrows). Nuclei were stained with DAPI (blue). At least two biological replicates were performed for each experiment depicted in A-D. Scale bars: 20 µm.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Inhibition, Recombinant, Staining

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: Suppression of STAT3 activity by pyrimethamine (PYR) inhibits VEGF-induced vascular permeability in zebrafish and mice. (A) Microangiography using 70 kDa Texas Red-dextran permeabilizing tracer (red) and 2000 kDa FITC-dextran intersegmental vessel marker (green) was performed on 3 dpf zebrafish without induced VEGF pretreated with DMSO ( n =6) or 25 μM PYR ( n =5) or 3 dpf zebrafish with induced VEGF pretreated with DMSO ( n =4) or 25 μM PYR ( n =9) for 3 days. Representative images shown were obtained using a Zeiss Apotome 2 microscope with a Fluar 5×/0.25 NA lens at RT. Scale bars: 50 μm. (B) The quantitative analysis of vascular permeability without VEGF stimulation or upon VEGF stimulation in zebrafish pretreated with DMSO or PYR. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. (C) Representative images of footpads from mice treated with vehicle or PYR following tail vein injection with 1% Evans Blue and footpad injection of VEGF (2.5 μg/ml) or PBS vehicle. (D) Quantitation of Evans Blue dye leakage in C57BL/6 WT mice treated with vehicle or PYR. n =9 mice in the vehicle group and n =7 mice in the PYR group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Multiple biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Activity Assay, Permeability, Marker, Microscopy, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: JAK2 phosphorylates STAT3 to transduce VEGF/VEGFR-2 signaling and promote vascular permeability. (A) To perform a STAT3 GST pull-down of VEGFR-2 and JAK2, lysates of HUVECs stimulated with serum for 30 min were used as prey. GST fusion protein STAT3 expressed in 293F cells was used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblotting. GST-STAT3 and GST were each detected using an anti-GST antibody. Three biological replicates were performed and depicted findings are representative. (B) JAK2 phosphorylates STAT3 in vitro . In vitro kinase assays were performed using purified human STAT3 protein and kinase active JAK2 protein. The results shown here are representative of two independent experiments. (C) Representative images of footpads from C57BL/6 WT mice treated with vehicle or JAK2 inhibitor AG490. Following tail vein injection with 1% Evans Blue dye, human VEGF-165 protein (2.5 μg/ml) or PBS vehicle was injected into the root of the footpad. After 30 min, the mice were euthanized and the footpads were excised. (D) Quantitation of Evans Blue dye leakage in C57BL/6 mice treated with vehicle or AG490. n =4 mice per group. Each mouse was injected with PBS in the right posterior footpad and VEGF in the left posterior footpad. Two biological replicates were performed and depicted findings are representative. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Transduction, Permeability, Negative Control, Binding Assay, SDS Page, Western Blot, In Vitro, Purification, Injection, Quantitation Assay

Journal: Disease Models & Mechanisms

Article Title: Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability

doi: 10.1242/dmm.049029

Figure Lengend Snippet: STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n =9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3 +/+ or VEGF; Stat3 −/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3 −/− ( n =5) compared to VEGF; Stat3 +/+ zebrafish ( n =7). Mean±s.e.m., unpaired, two-tailed Student's t -test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3 +/+ , n =3; Stat3 −/− , n =2) or 8 h following VEGF induction (Stat3 +/+ , n =4; Stat3 −/− , n =3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.

Article Snippet: Briefly, 10 µl of JAK2 protein diluted in kinase dilution buffer III (K23-09, Signal Chem) to a final concentration of 0.1 µg/ml was incubated with 3 µg purified STAT3 protein as well as 5 µl ATP (N0440S, New England Biolabs) for 30 min at 30°C.

Techniques: Permeability, Plasmid Preparation, Binding Assay, Mutagenesis, Luciferase, Transfection, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, Two Tailed Test, Expressing, Derivative 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: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

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: The in vitro kinase assay was performed using the Cyclin A2/Cdk1 Kinase Enzyme System (Promega, Madison, WI, USA), recombinant Cyclin B1/Cdk1 (SignalChem Biotech Inc., Richmond, BC, Canada), and ADP-Glo kinase assay (Promega), according to the manufacturers’ instructions.

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