Journal: Cell reports

Article Title: NMDAR-Activated PP1 Dephosphorylates GluN2B to Modulate NMDAR Synaptic Content

doi: 10.1016/j.celrep.2019.06.030

Figure Lengend Snippet: Levels of normalized GluN2B-pS1480 were analyzed by immunoblotting from cortical primary neurons after the indicated manipulations. Graphs represent mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001 using a Mann-Whitney U test (A and B) or Kruskal-Wallis H test (C-F). (A) Biotinylation experiment in DIV24-DIV28 neurons after induction of GluN2B-pS1480 dephosphorylation by NMDA incubation (50 μM for 10 min). Tubulin is shown as a control. M denotes marker. n = 5. (B) Isolation of the synaptic plasma membrane (SPM) fraction from DIV14-DIV21 neurons following NMDA treatment as before. Transferrin receptor (Tfr) is shown as a control. n = 5. (C) Synaptic, extrasynaptic, or global activation of NMDARs in DIV21-DIV28 neurons. pCreb and Creb are shown as controls. n = 3. (D) Incubation of DIV14-DIV17 neurons with phosphatase inhibitors for 45 min: fostriecin for PP2A/4 (Fos; 1 μM); FK506 for PP2B (FK; 1 μM); okadaic acid for PP2A at 20 nM and PP2A/PP1 at 50 nM (OA; 20 or 50 nM); and calyculin A for PP1/PP2A (Cal A; 100 nM). n = 10. (E) Pre-incubation of DIV14-DIV21 neurons with 100 nM Cal A for 45 min before NMDA treatment as before. p = 0.66 (Cal A versus Cal A + NMDA). n = 14. (F) Lentiviral transduction of the dominant negative forms of GFP-PP1 α and γ1 (PP1α/γ1 D95N) and GFP (as a control) in DIV14-DIV17 neurons for 7–10 days. GluN2B-pS1480 dephosphorylation was induced with NMDA as before. p > 0.99 (PP1α) and p = 0.78 (PP1γ1). n = 6. See also and .

Article Snippet: Okadaic Acid , Tocris , Cat#1136; CAS: 78111-17-8.

Techniques: Western Blot, MANN-WHITNEY, De-Phosphorylation Assay, Incubation, Control, Marker, Isolation, Clinical Proteomics, Membrane, Activation Assay, Transduction, Dominant Negative Mutation

Journal: Cell reports

Article Title: NMDAR-Activated PP1 Dephosphorylates GluN2B to Modulate NMDAR Synaptic Content

doi: 10.1016/j.celrep.2019.06.030

Figure Lengend Snippet:

Article Snippet: Okadaic Acid , Tocris , Cat#1136; CAS: 78111-17-8.

Techniques: Recombinant, Software, Imaging

Journal: The Biochemical journal

Article Title: Enhancement of mDia2 activity by Rho-kinase-dependent phosphorylation of the diaphanous autoregulatory domain

doi: 10.1042/BJ20101700

Figure Lengend Snippet: (A) Schematic diagram of phosphorylation site conservation near the mDia2 DAD basic region. Note that similar sites are not found in Dia1 or Dia3. (B) Left-hand panel, FLAG–mDia2 and Myc–ROCK1 were co-expressed in HeLa cells. FLAG immunoprecipitates (IP) were run on an SDS/PAGE gel, transferred on to nitrocellulose, and probed for Myc. Input controls represent 10 % of immunoprecipitated fractions. Ev, empty vector. Right-hand panel, endogenous ROCK1 immunoprecipitates were subjected to Western blotting (IB) for mDia2. (C) GST–mDia2 DAD variants containing the indicated phosphorylation mutations were incubated in vitro with constitutively active ROCKΔ3 and 10 μCi of [γ-32P]ATP. After removing unincorporated 32P, reactions were run on an SDS/PAGE gel and exposed to film. The ROCK inhibitor Y-27632 (Y; 10 μM) was added to some reactions. (D) Cos-7 cells were transfected with the indicated mDia2 DAD variant along with ROCK1Δ3. Following [32P]Pi labelling for 2 h, the mDia2 peptides were immunoprecipitated and analysed by autoradiography. Okadaic acid was added to some cells ± pretreatment with Y-27632 (10 μM). (E) Cos-7 cells were transfected with the indicated full-length mDia2 variant along with ROCK1Δ3. Three independent phosphorylation experiments were quantified by densitometry. *P < 0.05 compared with wild-type minus ROCK1Δ3, **P < 0.05 compared with wild-type plus ROCK1Δ3. (F) Same as in (E), except that cells were transfected with the indicated glutamate variants of mDia2. (G) HeLa cells (top) or HeLa cells transfected with the wild-type or DAD 2A mDia2 variant (bottom) were treated with Y-27632 (or vehicle) for 1 h and then with calyculin A (or vehicle) for an additional 1 h. RIPA lysates were run on an SDS/PAGE gel and subjected to Western blotting analysis using anti-mDia2 or anti-FLAG antibodies. Some lysates were treated with SAP before loading. FL, full-length; WT/Wt, wild-type.

Article Snippet: Chemical reagents used were as follows: Y-27632, calyculin A and latrunculin B (Calbiochem); jasplakinolide (EMD chemicals) and okadaic acid (MP Biomedicals).

Techniques: SDS Page, Immunoprecipitation, Plasmid Preparation, Western Blot, Incubation, In Vitro, Transfection, Variant Assay, Autoradiography

Journal: Journal of Marine Science and Engineering

Article Title: A Protein Phosphatase 2A-Based Assay to Detect Okadaic Acids and Microcystins

doi: 10.3390/jmse12020244

Figure Lengend Snippet: Figure 7. Calibration curve using the net absorbance of OA standard solutions. The PP2A inhibition assay was performed using OA standard solutions (0, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0, and 10.0 ng/mL) prepared from OA CRM (CRM-OA-d, NRC Canada) according to the method described in the reference [16]. Each point in the calibration curve represents the mean (n = 3) with an RSD of 2.5%.

Article Snippet: The PP2A inhibition assay was performed using OA standard solutions (0, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0, and 10.0 ng/mL) pre- pared from OA CRM (CRM-OA-d, NRC Canada) according to the method described in the reference [16].

Techniques: Inhibition