Journal: Children

Article Title: Thirteen New Patients of PPP2R5D Gene Mutation and the Fine Profile of Genotype–Phenotype Correlation Unraveling the Pathogenic Mechanism Underlying Macrocephaly Phenotype

doi: 10.3390/children11080897

Figure Lengend Snippet: Protein expression levels of PPP2R5D and its interaction with the PPP2CA and PPP2R1A. ( A ) HA-tagged PPP2R5D WT/variants were transfected in 293T cells, protein expression was detected through Western blot. A 66 kDa band was detected in all missense variants. As for frameshift variant H436Mfs*3, a ~50 kDa band was detected and it was significantly weaker. ( B ) HA-tagged PPP2R5D WT/variants were purified through HA pull down, the interaction with endogenous PPP2CA and PPP2R1A were detected through Western blot. No band of PPP2CA and PPP2R1A was detected in W207R, D251H, D251Y and H463Mfs*3. A weaker band of PPP2CA and PPP2R1A was detected in E198K, E200K, L203P and Q211P.

Article Snippet: The primary antibodies are listed as below: PPP2R5D (Abcam, #ab188323, Cambridge, UK), Vinculin (Cell Signaling Technology, #13901), PPP2R1A (NOVUS, #NBP2-19907, St. Louis, MO, USA), and PPP2CA (Cell Signaling Technology, #2308).

Techniques: Expressing, Transfection, Western Blot, Variant Assay, Purification

Journal: Children

Article Title: Thirteen New Patients of PPP2R5D Gene Mutation and the Fine Profile of Genotype–Phenotype Correlation Unraveling the Pathogenic Mechanism Underlying Macrocephaly Phenotype

doi: 10.3390/children11080897

Figure Lengend Snippet: Pathogenicity prediction of PPP2R5D and analysis of residue characterization and phenotype. ( A ) Pathogenicity prediction of PPP2R5D by AM, the yellow bar represents conserved region 1, 2, and 3. Observed pathogenic variants were represented by red dots. ( B ) Protein 3D structure of PP2A holoenzyme. PPP2R5D protein was colored by scores predicted by AM, from green to red, low to high. PPP2R1A and PPP2CA were colored grey and darker gray. Amino acids affected by pathogenic missense variants of PPP2R5D gene were labeled on the protein. ( C ) Pathogenicity prediction by AM of PPP2CA and PPP2R1A. ( D ) Two-sided Fisher’s exact test of phenotypes and characterization of residues. Circles represent the OR, and the horizontal bars represent 95% CI. Red circles mean that the features in the y axis were enriched in patients with the phenotype, green means the opposite.

Article Snippet: The primary antibodies are listed as below: PPP2R5D (Abcam, #ab188323, Cambridge, UK), Vinculin (Cell Signaling Technology, #13901), PPP2R1A (NOVUS, #NBP2-19907, St. Louis, MO, USA), and PPP2CA (Cell Signaling Technology, #2308).

Techniques: Residue, Labeling

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Clinicopathological effects of protein phosphatase 2, regulatory subunit A, alpha mutations in gastrointestinal stromal tumors.

doi: 10.1038/modpathol.2016.138

Figure Lengend Snippet: Figure 1 Examples of PPP2R1A mutations. The left two panels (a and c) show mutations detected in exon 5 of case #6, and the right two panels (b and d) show mutations detected in exon 6 of cases #10 and #15. These mutations were tumor-specific. (a and b) DNA derived from non-tumor tissue. (c) DNA derived from tumor tissue samples was examined and a PPP2R1A variant at codon 195 GTG4ATG (V195M) was detected. (d) DNA from tumor tissue was examined and a PPP2R1A variant at codon 238 GAG4AAG (E238K) was detected.

Article Snippet: A plasmid encoding human PPP2R1A (Origene) was used to generate constructs, which were subcloned into pGEM. cDNA encoding the PPP2R1A Val201Ala (PPP2R1A-T602C) and Glu238Lys (PPP2R1AG712A) mutants was generated using the QuickChange II Site-Directed Mutagenesis kit (Agilent Technologies), and these constructs were subcloned into pCX4bleo.

Techniques: Derivative Assay, Variant Assay

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Clinicopathological effects of protein phosphatase 2, regulatory subunit A, alpha mutations in gastrointestinal stromal tumors.

doi: 10.1038/modpathol.2016.138

Figure Lengend Snippet: Figure 2 Prognostic impact of PPP2R1A mutation in GISTs. Both overall survival (a) and disease-free survival (b) were significantly different between the mutation-positive and mutation-negative cases (overall survival Po0.05, disease-free survival Po0.05).

Article Snippet: A plasmid encoding human PPP2R1A (Origene) was used to generate constructs, which were subcloned into pGEM. cDNA encoding the PPP2R1A Val201Ala (PPP2R1A-T602C) and Glu238Lys (PPP2R1AG712A) mutants was generated using the QuickChange II Site-Directed Mutagenesis kit (Agilent Technologies), and these constructs were subcloned into pCX4bleo.

Techniques: Mutagenesis

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Clinicopathological effects of protein phosphatase 2, regulatory subunit A, alpha mutations in gastrointestinal stromal tumors.

doi: 10.1038/modpathol.2016.138

Figure Lengend Snippet: Figure 3 (a) Human phospho-kinase array analysis. Levels of phospho-kinases were assessed using a horseradish peroxidase- conjugated phospho-kinase antibody, which was followed by chemiluminescence detection. In T1-WT cells, the levels of phosphorylated Akt1/2/3, ERK1/2, and WNK1 were significantly reduced. Both T1-T602C and T1-G712A cells had significantly higher levels of phosphorylated Akt1/2/3 and WNK1. The level of phosphorylated ERK1/2 increased in T1-G712A cells and decreased in T1-T602C cells. *Po0.05 compared with cells expressing PPP2R1A-WT. (b) Evaluation of in vitro cell growth. For each experiment, 1.0× 106 cells were cultured in RPMI and counted at 24, 72, and 120 h. *Po0.05 compared with cells expressing GFP; NS, not significant compared with cells expressing GFP.

Article Snippet: A plasmid encoding human PPP2R1A (Origene) was used to generate constructs, which were subcloned into pGEM. cDNA encoding the PPP2R1A Val201Ala (PPP2R1A-T602C) and Glu238Lys (PPP2R1AG712A) mutants was generated using the QuickChange II Site-Directed Mutagenesis kit (Agilent Technologies), and these constructs were subcloned into pCX4bleo.

Techniques: Expressing, In Vitro, Cell Culture

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Clinicopathological effects of protein phosphatase 2, regulatory subunit A, alpha mutations in gastrointestinal stromal tumors.

doi: 10.1038/modpathol.2016.138

Figure Lengend Snippet: Figure 4 c-kit phosphorylation status according to the transduc- tion of mutant PPP2R1A in T1 cells. In T1 cells transduced with mutant PPP2R1A, phosphorylation at Tyr721 was increased compared with those transduced with wild-type PPP2R1A or GFP. The same phenomenon was observed regarding phosphor- ylation of Tyr568/570, however, this trend was weaker than Tyr721. On the other hand, in wild-type-transduced cells, phosphorylation was decreased compared with T1-GFP.

Article Snippet: A plasmid encoding human PPP2R1A (Origene) was used to generate constructs, which were subcloned into pGEM. cDNA encoding the PPP2R1A Val201Ala (PPP2R1A-T602C) and Glu238Lys (PPP2R1AG712A) mutants was generated using the QuickChange II Site-Directed Mutagenesis kit (Agilent Technologies), and these constructs were subcloned into pCX4bleo.

Techniques: Phospho-proteomics, Mutagenesis, Transduction

Journal: PloS one

Article Title: PR65A phosphorylation regulates PP2A complex signaling.

doi: 10.1371/journal.pone.0085000

Figure Lengend Snippet: Figure 1. Identification of phosphorylated amino acids in cardiac PR65A. MALDI/TOF spectra for PR65A extracted from Dahl R rat hearts show that S303 (Panel A), T268 (Panel B), and S314 (Panel C) are phosphorylated. Methods: Phosphoproteins from control Dahl R rats were separated in 2D-DIGE. The protein spot corresponding to PR65A was excised from the gel, subjected to tryptic digestion and phosphopeptide enrichment using Supel-Tips (Sigma-Aldrich, Inc). The phosphopeptides were spotted on an eMALDI plate followed by mass spectrometry and database search. The spectra of all peptides were manually evaluated for the loss of phosphate which is shown in red circles and the mass and sequence of the peptides are shown. X-axis represents mass and Y axis represents intensity (see General Experimental Methods for details). doi:10.1371/journal.pone.0085000.g001

Article Snippet: PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e85000 PR65a constructs Full-length PR65a DNA construct (Origene Inc) was used to introduce S/T to A (T268A, S303A, and S314A) and S/T to E mutations (T268E, S303E, and S314E) by site-directed mutagenesis using the QuikChangeTM mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols.

Techniques: Control, Phospho-proteomics, Mass Spectrometry, Sequencing

Journal: PloS one

Article Title: PR65A phosphorylation regulates PP2A complex signaling.

doi: 10.1371/journal.pone.0085000

Figure Lengend Snippet: Figure 2. PP2A phosphatase activity and interaction of PR65A with PP2Ac is reduced by PR65A phosphorylation. Panel 2A: PP2A activity in immunoprecipitates of protein extracts from HEK cells transfected with recombinant N-PR65A vs. P-PR65A. N = 3; * P,0.01. Panel 2B: HEK cells were transfected with either N-PR65A or P-PR65A constructs immunoprecipitated with anti-PR65A polyclonal antibody (see General Experimental Methods) followed by Western blotting with anti-PP2Ac Ab and PR65A Ab. Panel 2C: HEK cells were transfected with either N-PR65A or P-PR65A constructs and protein extracts were immunoblotted with anti-PP2Ac Ab. (n = 3). doi:10.1371/journal.pone.0085000.g002

Article Snippet: PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e85000 PR65a constructs Full-length PR65a DNA construct (Origene Inc) was used to introduce S/T to A (T268A, S303A, and S314A) and S/T to E mutations (T268E, S303E, and S314E) by site-directed mutagenesis using the QuikChangeTM mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols.

Techniques: Activity Assay, Phospho-proteomics, Transfection, Recombinant, Construct, Immunoprecipitation, Western Blot

Journal: PloS one

Article Title: PR65A phosphorylation regulates PP2A complex signaling.

doi: 10.1371/journal.pone.0085000

Figure Lengend Snippet: Figure 3. 2D-DIGE Analysis of phosphoproteins from N-PR65A (green) vs. P-PR65A (red) transfected HEK cells. Panel A: Phospho- enriched protein samples from cells transfected with N-PR65A vs. P-PR65A were differentially labeled with Cydyes (N-PR65A = Cy5 red, P-PR65A = Cy2 green) and subjected to 2D-DIGE followed by phosphoprotein profiling (see General Methods). Molecular weight markers (kDa) are displayed to the left of each gel and pH markers are displayed underneath each gel. The spots are numbered according to size and PI. Spots 8, 23, 25, 28, 37, 38, 65, and 88 were identified. Panel B: Validation of increased phosphorylation of elongation factor-1 alpha by recombinant P-PR65A. Protein extracts from HEK cells transfected with N-PR65A and P-PR65A were immunoprecipitated with anti-phospho serine/threonine Ab followed by Western blotting with anti-rabbit monoclonal antibody against elongation factor 1 alpha. n = 3; * P,0.01. doi:10.1371/journal.pone.0085000.g003

Article Snippet: PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e85000 PR65a constructs Full-length PR65a DNA construct (Origene Inc) was used to introduce S/T to A (T268A, S303A, and S314A) and S/T to E mutations (T268E, S303E, and S314E) by site-directed mutagenesis using the QuikChangeTM mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols.

Techniques: Transfection, Labeling, Molecular Weight, Biomarker Discovery, Phospho-proteomics, Recombinant, Immunoprecipitation, Western Blot

Journal: PloS one

Article Title: PR65A phosphorylation regulates PP2A complex signaling.

doi: 10.1371/journal.pone.0085000

Figure Lengend Snippet: Figure 4. Reduced expression of phosphorylated PR65A and greater PP2A activity in systolic failing hearts. Panel A: Protein extracts from control (Dahl R) and (Dahl S) rat hearts were immunoprecipitated with monoclonal anti-phospho serine/threonine antibodies followed by Western blotting with monoclonal Ab against PR65A. n = 3 P,0.05. As a control protein extracts from control (Dahl R) and (Dahl S) rat hearts were subjected to SDS-PAGE followed by Western blotting with Ab against PR65A. Panel B: PP2A phosphatase activity in systolic failing hearts. PP2A activity in protein extracts from left ventricular walls of systolic failing (Dahl S) and control (Dahl R) rats, N = 4; *P,0.01. Panel C: Western blotting of protein extracts from control and failing rat hearts with Ab against PP2A catalytic subunit (n = 4). doi:10.1371/journal.pone.0085000.g004

Article Snippet: PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e85000 PR65a constructs Full-length PR65a DNA construct (Origene Inc) was used to introduce S/T to A (T268A, S303A, and S314A) and S/T to E mutations (T268E, S303E, and S314E) by site-directed mutagenesis using the QuikChangeTM mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols.

Techniques: Expressing, Activity Assay, Control, Immunoprecipitation, Western Blot, SDS Page

Journal: PloS one

Article Title: PR65A phosphorylation regulates PP2A complex signaling.

doi: 10.1371/journal.pone.0085000

Figure Lengend Snippet: Figure 5. Proposed defect in PP2A holoenzyme assembly by phosphorylation of PR65A. (A) Phosphorylation and modeled changes in HEAT repeats 7, 8, 9 of the A-subunit. The A-subunit from the PR70 holoenzyme (left, purple), the modeled structure of the phosphorylated A subunit (middle, light green), and their overlay (right) are shown. Residues Thr268, Ser303, Ser314, and their phosphorylation counterparts are shown in cylinder and colored by atom type. (B) Alignment of the PR70 and B9c1 holoenzymes and the model of the phosphorylated A subunit by HEAT repeats 11–15. The A subunits are in ribbon. PP2Ac and the PR70 regulatory subunit are in space fill. The A subunits from the PR70 and B9c1 holoenzymes and the model of the phosphorylated A subunit are colored purple, yellow, and light green, respectively. doi:10.1371/journal.pone.0085000.g005

Article Snippet: PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e85000 PR65a constructs Full-length PR65a DNA construct (Origene Inc) was used to introduce S/T to A (T268A, S303A, and S314A) and S/T to E mutations (T268E, S303E, and S314E) by site-directed mutagenesis using the QuikChangeTM mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols.

Techniques: Phospho-proteomics

Journal: Reproductive toxicology (Elmsford, N.Y.)

Article Title: Determinants of Orofacial Clefting I: Effects of 5-Aza-2′-deoxycytidine on Cellular Processes and Gene Expression during Development of the First Branchial Arch

doi: 10.1016/j.reprotox.2016.11.016

Figure Lengend Snippet: Genes Differentially Expressed within 1 st Branchial Arches Following 12 hour in utero Exposure to 1 mg/kg AzaD on GD 9.5.

Article Snippet: Ppp2r1a , Protein phosphatase 2, regulatory subunit A, alpha , Mm00772799_m1 , 2.04 , 0.03.

Techniques: In Utero, TaqMan Assay, Binding Assay, Membrane, Histone Deacetylase Assay, Translocation Assay, Ubiquitin Proteomics

Journal: Pharmacology Research & Perspectives

Article Title: Stathmin dynamics modulate the activity of eribulin in breast cancer cells

doi: 10.1002/prp2.786

Figure Lengend Snippet: Effects of a PP2A inhibitor and activator on eribulin‐induced phosphorylation of stathmin. MCF7 and MDA‐MB‐231 cells were treated for 1 h with a PP2A inhibitor (okadaic acid: OA) or activator (FTY‐720: FTY), and then stimulated for 24 h with 10 nM eribulin. Cell lysates were subjected to immunoblot analysis to detect p‐stathmin and stathmin. GAPDH served as a loading control. Representative data from three independent experiments in duplicate are shown

Article Snippet: Briefly, recombinant PP2A (PP2Aα/PPP2R1A Complex, SignalChem), dissolved in a buffer comprising 50 mM Tris‐HCl (pH 8.4), 10 mM MgCl 2 , 0.5 M EDTA, 1 mM DTT, and 0.05 mg/ml BSA, was incubated for 5 min with 0.1–10 nM okadaic acid, eribulin, or paclitaxel.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Pharmacology Research & Perspectives

Article Title: Stathmin dynamics modulate the activity of eribulin in breast cancer cells

doi: 10.1002/prp2.786

Figure Lengend Snippet: Effects of eribulin on PP2A activity and expression. (A) Recombinant PP2A was incubated for 5 min with various concentrations of okadaic acid (OA), eribulin, or paclitaxel (PTX), followed by p‐nitrophenyl phosphate (pNPP) for 1 h. Absorbance at 405 nm was measured in a microplate reader. (B) MCF7 and MDA‐MB‐231 cells were treated with eribulin for 24 h. Total RNA was analyzed by real‐time RT‐PCR to determine expression of SET , PME ‐ 1 , and CIP2A . (C) Cells were stimulated with eribulin (3 or 10 nM) for 24 h. Cell lysates were subjected to immunoblot analysis to detect PP2A subunits PP2Aa, PP2Ab, and PP2Ac. GAPDH served as a loading control. (D) Expression of PPP2R1A and PPP2CA was analyzed by real‐time RT‐PCR

Article Snippet: Briefly, recombinant PP2A (PP2Aα/PPP2R1A Complex, SignalChem), dissolved in a buffer comprising 50 mM Tris‐HCl (pH 8.4), 10 mM MgCl 2 , 0.5 M EDTA, 1 mM DTT, and 0.05 mg/ml BSA, was incubated for 5 min with 0.1–10 nM okadaic acid, eribulin, or paclitaxel.

Techniques: Activity Assay, Expressing, Recombinant, Incubation, Quantitative RT-PCR, Western Blot, Control

Journal: Pharmacology Research & Perspectives

Article Title: Stathmin dynamics modulate the activity of eribulin in breast cancer cells

doi: 10.1002/prp2.786

Figure Lengend Snippet: Proposed mechanism by which eribulin exerts antiproliferative effects through modulating stathmin dynamics in breast cancer cell. Eribulin phosphorylates stathmin by activating the PKA and CaMKII pathways, and by downregulating PP2A level in breast cancer cells. Overexpression of stathmin potentiates the antiproliferative effects of eribulin

Article Snippet: Briefly, recombinant PP2A (PP2Aα/PPP2R1A Complex, SignalChem), dissolved in a buffer comprising 50 mM Tris‐HCl (pH 8.4), 10 mM MgCl 2 , 0.5 M EDTA, 1 mM DTT, and 0.05 mg/ml BSA, was incubated for 5 min with 0.1–10 nM okadaic acid, eribulin, or paclitaxel.

Techniques: Over Expression

Journal: Leukemia

Article Title: Low Expression Of PP2A Regulatory Subunit B55α Is Associated With T308 Phosphorylation Of AKT And Shorter Complete Remission Duration In Acute Myeloid Leukemia Patients

doi: 10.1038/leu.2011.146

Figure Lengend Snippet: List of PP2A subunit genes and ABL1 control analyzed using Real-Time PCR with Applied Biosystems (ABI) Taqman assays.

Article Snippet: PP2A A α , PPP2R1A , 19q13.41 , Hs00204426_m1.

Techniques: Control, Real-time Polymerase Chain Reaction

Journal: Leukemia

Article Title: Low Expression Of PP2A Regulatory Subunit B55α Is Associated With T308 Phosphorylation Of AKT And Shorter Complete Remission Duration In Acute Myeloid Leukemia Patients

doi: 10.1038/leu.2011.146

Figure Lengend Snippet: Real Time PCR was performed as described in “Materials and Methods”. Expression of PPP2R1A (A α), PPP2R1B (A β), PPP2R2A (B55 α), PPP2CA (C α), and PPP2CB(C β) genes are presented as relative to 100 copies of ABL1for CD34+ cells derived from bone marrow from normal donors (N = 6) and blast cells derived from AML patients (N= 75). The p values were derived as described in “Materials and Methods”. Statistically significant differences from expression in normal bone marrow cells (p < 0.05) are marked with *.

Article Snippet: PP2A A α , PPP2R1A , 19q13.41 , Hs00204426_m1.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Derivative Assay