pp2a catalytic subunit expression plasmid  (OriGene)

Journal: British journal of pharmacology

Article Title: Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

doi: 10.1111/bph.16503

Figure Lengend Snippet: FIGURE 8 Effects of inhibitors of AMPK and PP2A on decreased 5-HT receptor expression on astrocytes induced by exposure to clozapine and L-BAIBA. Cultured astrocytes were incubated in fDMEM containing 3 μM clozapine (CLZ), 3 μM clozapine plus 10 μM dorsomorphin (CLZ + Dor), 3 μM clozapine plus 3 μM LB-100 (CLZ + LB) or 30 μM L-BAIBA (BAIBA), 30 μM L-BAIBA plus 10 μM dorsomorphin (BAIBA + Dor) or 30 μM L-BAIBA plus 3 μM LB-100 (BAIBA + LB) for 14 days. In the upper-side histograms, ordinate: mean ± SD (n = 6) of the relative levels of 5-HT1A (a and d), 5-HT2A (b and e) and 5-HT7 receptors (c and f) relative to control in the plasma membrane fraction of cultured astrocytes. *P <0.05 relative to 3 μM clozapine alone or 30 μM L-BAIBA alone using one-way ANOVA with Scheffe's post hoc test. The lower panel indicates pseudo-gel images of capillary immunoblotting. F values of the effects of clozapine, dorsomorphin and LB-100 on 5-HT1A, 5-HT2A and 5-HT7 receptors in plasma membrane fraction were [F(2,15) = 23.9 (P <0.05)], [F(2,15) = 19.4 (P <0.05)] and [F(2,15) = 65.6 (P <0.05)], respectively. F values of effects of L-BAIBA, dorsomorphin and LB-100 on 5-HT1A, 5-HT2A and 5-HT7 receptors in the plasma membrane fraction were [F(2,15) = 15.9 (P <0.05)], [F(2,15) = 28.7 (P <0.05)] and [F(2,15) = 12.1 (P <0.05)], respectively.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_ 10001915, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (8475, RRID:AB_3102029, 1:50, Cell Signalling Technology, Danvers, MA, USA), γ-actin (GTX101794, RRID:AB_2036153, 1:100, GeneTex, Irvine, CA, USA), connexin43 (C6219, RRID:AB_476857, 1:100, Sigma-Aldrich), PP2A (AF1653, RRID:AB_354917, 1 μg ml 1, R&D systems, Minneapolis, MN, USA), phosphorylated-PP2A (pPP2A: AF3989, RRID:AB_2169636, 10 μg ml 1, R&D Systems), Akt (AF1775, RRID:AB_354982, 1 μg ml 1, R&D Systems), phosphorylated-Akt (pAkt: AF887, RRID:AB_355685, 5 μg ml 1, R&D Systems), AMPKα (2603, RRID:AB_490795, 1:50, Cell Signalling Technology, Danvers, MA, USA), phosphorylated-AMPKα (pAMPK: 2535, RRID:AB_331250, 1:50, Cell Signalling Technology), 5-HT1A receptor (5-HT1A-R: NBP2–21590, RRID:AB_3102030, 1:100, Novus Biologicals, Littleton, CO, USA), 5-HT2A receptor (5-HT2A-R: LS-C172270, RRID:AB_3102031, 1:50, Lifespan Biosciences, WA, USA), 5-HT7 receptor (5-HT7-R, NB100-56352, RRID:AB_2264237, 1:50, Novus Biologicals) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio).

Techniques: Expressing, Cell Culture, Incubation, Control, Clinical Proteomics, Membrane, Western Blot

Journal: British journal of pharmacology

Article Title: Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

doi: 10.1111/bph.16503

Figure Lengend Snippet: FIGURE 9 Interactions among clozapine, L-BAIBA and AMPK inhibitors on PP2A without clozapine, with 3 μM clozapine alone (CLZ), 3 μM clozapine plus 10 μM dorsomorphin (CLZ + Dor) (a), or without 30 μM L-BAIBA, with 30 μM L-BAIBA alone (BAIBA), or 30 μM L-BAIBA plus 10 μM dorsomorphin (BAIBA + Dor) (b) for 14 days. In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pPP2A relative to control. *P <0.05 relative to control (clozapine or L-BAIBA free) by one-way ANOVA with Scheffe's post hoc test. The lower panel indicates pseudo-gel images of capillary immunoblotting. The F values for the effects of clozapine and dorsomorphin on pPP2A were F(2,15) = 22.9 (P <0.05). The F values for the effects of L-BAIBA and dorsomorphin on pPP2A were [F(2,15) = 20.9 (P <0.05)].

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_ 10001915, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (8475, RRID:AB_3102029, 1:50, Cell Signalling Technology, Danvers, MA, USA), γ-actin (GTX101794, RRID:AB_2036153, 1:100, GeneTex, Irvine, CA, USA), connexin43 (C6219, RRID:AB_476857, 1:100, Sigma-Aldrich), PP2A (AF1653, RRID:AB_354917, 1 μg ml 1, R&D systems, Minneapolis, MN, USA), phosphorylated-PP2A (pPP2A: AF3989, RRID:AB_2169636, 10 μg ml 1, R&D Systems), Akt (AF1775, RRID:AB_354982, 1 μg ml 1, R&D Systems), phosphorylated-Akt (pAkt: AF887, RRID:AB_355685, 5 μg ml 1, R&D Systems), AMPKα (2603, RRID:AB_490795, 1:50, Cell Signalling Technology, Danvers, MA, USA), phosphorylated-AMPKα (pAMPK: 2535, RRID:AB_331250, 1:50, Cell Signalling Technology), 5-HT1A receptor (5-HT1A-R: NBP2–21590, RRID:AB_3102030, 1:100, Novus Biologicals, Littleton, CO, USA), 5-HT2A receptor (5-HT2A-R: LS-C172270, RRID:AB_3102031, 1:50, Lifespan Biosciences, WA, USA), 5-HT7 receptor (5-HT7-R, NB100-56352, RRID:AB_2264237, 1:50, Novus Biologicals) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio).

Techniques: Control, Western Blot

Journal: British journal of pharmacology

Article Title: Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

doi: 10.1111/bph.16503

Figure Lengend Snippet: FIGURE 11 Proposed hypothesis of pathophysiology of clozapine discontinuation syndrome. During clozapine exposure, clozapine increases L-BAIBA and Akt signalling, but inhibits 5-HT receptors (and histamine H1 receptors) (Carli et al., 2021; Fukuyama et al., 2023a, 2023b, 2023c). Inhibition of 5-HT2 receptors decreases IP3 synthesis, leading to attenuation of IP3 receptors (Okada et al., 2005). Increased intracellular calcium levels activate ATP synthase activity (Carli et al., 2021; de Brito & Scorrano, 2010). Clozapine also increases L-BAIBA synthesis, which activates AMPK signalling via possibly MRGPRD activation (Fukuyama et al., 2023a, 2023c; Kitase et al., 2018; Minato et al., 2022). Therefore, clozapine exposure activates AMPK signalling through 5-HT2 receptor inhibition and increases L-BAIBA. Clozapine-induced activation of hypothalamic AMPK signalling plays important roles in weight-gain and metabolic complication of clozapine (Carli et al., 2021). Activation of AMPK signalling suppresses PP2A signalling, resulting in decreasing 5-HT receptors in the plasma membrane via suppression of receptor recycling (Raote et al., 2013; Tsao et al., 2001). On the contrary, connexin43 trafficking is enhanced via activation of Akt signalling, resulting in increased hemichannels containing connexin43 in the astroglial plasma membrane (Fukuyama et al., 2023a; Okada, 2022; Smith et al., 2014). Astroglial D-serine release through hemichannels increases during clozapine exposure. Clozapine discontinuation, intracellular L-BAIBA level and signallings AMPK, Akt and PP2A immediately recovered. PP2A signalling immediately recovered during subsequent clozapine discontinuation, in comparison clozapine exposure transiently increased 5-HT receptor expression (until 7 days of discontinuation), resulting in transiently increased intracellular IP3 levels. PP2A also activates phosphatidylinositol turnover (Zhong et al., 2008). Connexin43 expression also recovered after clozapine discontinuation, but increased astroglial D-serine release persisted until 7 days after clozapine discontinuation due to transiently activation of IP3 receptors via transiently increased IP3 level.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_ 10001915, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (8475, RRID:AB_3102029, 1:50, Cell Signalling Technology, Danvers, MA, USA), γ-actin (GTX101794, RRID:AB_2036153, 1:100, GeneTex, Irvine, CA, USA), connexin43 (C6219, RRID:AB_476857, 1:100, Sigma-Aldrich), PP2A (AF1653, RRID:AB_354917, 1 μg ml 1, R&D systems, Minneapolis, MN, USA), phosphorylated-PP2A (pPP2A: AF3989, RRID:AB_2169636, 10 μg ml 1, R&D Systems), Akt (AF1775, RRID:AB_354982, 1 μg ml 1, R&D Systems), phosphorylated-Akt (pAkt: AF887, RRID:AB_355685, 5 μg ml 1, R&D Systems), AMPKα (2603, RRID:AB_490795, 1:50, Cell Signalling Technology, Danvers, MA, USA), phosphorylated-AMPKα (pAMPK: 2535, RRID:AB_331250, 1:50, Cell Signalling Technology), 5-HT1A receptor (5-HT1A-R: NBP2–21590, RRID:AB_3102030, 1:100, Novus Biologicals, Littleton, CO, USA), 5-HT2A receptor (5-HT2A-R: LS-C172270, RRID:AB_3102031, 1:50, Lifespan Biosciences, WA, USA), 5-HT7 receptor (5-HT7-R, NB100-56352, RRID:AB_2264237, 1:50, Novus Biologicals) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio).

Techniques: Inhibition, Activity Assay, Activation Assay, Clinical Proteomics, Membrane, Comparison, Expressing

Journal: BMC medicine

Article Title: LASS2 enhances chemosensitivity to cisplatin by inhibiting PP2A-mediated β-catenin dephosphorylation in a subset of stem-like bladder cancer cells.

doi: 10.1186/s12916-023-03243-5

Figure Lengend Snippet: Fig. 7 Proposed model for the role of LASS2 in cisplatin resistance in bladder cancer. Left: Loss of LASS2 leads to activation of PP2A by increasing C18-ceramide, while PP2A dephosphorylates β-catenin to promote nuclear translocation and activate β-catenin/TCF4 signaling. Right: LASS2 decreases PP2A activity by reducing C18-ceramide and dissociating PP2A from β-catenin, leading to the degradation of phospho-β-catenin

Article Snippet: The LASS2 ORF expression plasmid (#RG215300) and PP2A catalytic subunit expression plasmid (#SC321401) with the pCMV6-Ac-GFP vector were obtained from OriGene. siRNAs for GSK3β (sc-35525) and CK1α (sc29912) were obtained from Santa Cruz.

Techniques: Activation Assay, Translocation Assay, Activity Assay

Journal: Nature Communications

Article Title: Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells

doi: 10.1038/s41467-023-44109-x

Figure Lengend Snippet: A PP2A activity measured after immunoprecipitation from RBC incubated with vehicle and 1 µM Sph for 30 min (n = 5 each). B Glucose uptake and ( C ) GLUT4 cell surface localization in sphingosine-loaded RBC in the absence or presence of 2 nM of the PP2A inhibitor okadaic acid (n = 6 each). D PP2A activity measured in SphK1 +/+ and SphK1 –/– RBC (n = 3/4), and ( E ) PP2A activity in Mfsd2b +/+ and Mfsd2b –/– RBC (n = 3 each). F PP2A activity of the holoenzyme immunoprecipitated from C57Bl6 RBC lysates and incubated consecutively with vehicle, sphingosine, S1P, FTY720, FTY720-P and C6 ceramide in the indicated concentrations (n = 10/6/8/6/6/4/4). G PP2A activity of human recombinant PP2AC catalytic subunit in the presence of the same substances as in ( E ) (n = 7/3/7/4/4/4). The ‘n’ in the ( A – G ) refers to individual mice. Data are presented as mean ± sd and tested with paired two-tailed t test ( A ), two-way ANOVA ( B , C ), unpaired t test ( D , E ), and one-way ANOVA ( F , G ); ns= not significant; p* < 0.05; P** < 0.01; P*** < 0.001; P**** < 0.0001.

Article Snippet: In the case of human recombinant PP2A catalytic subunit (Cayman Chemical, Ann Arbor, USA) 0.58 mU in 100 μl were incubated for 10 min with all substances.

Techniques: Activity Assay, Immunoprecipitation, Incubation, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells

doi: 10.1038/s41467-023-44109-x

Figure Lengend Snippet: A Intracellular S1P levels and ( B ) corresponding S1P efflux in human RBC after incubation without or with 1 µM Sph for 30 min followed by exposure or not to 1% BSA for 30 min (n = 6 each). C Glucose uptake rate in human RBC treated as in ( A ) (n = 9). D PP2A activity of human RBC treated with 1 µM Sph or vehicle (n = 6 each). E Representative flow cytometry histograms of surface GLUT1 as measured by H RBD EGFP binding after incubation with 1 µM Sph or vehicle for 30 min. F Quantification of GLUT1 fluorescence from human RBC treated with 1 µM Sph or vehicle (n = 4). MFI (mean fluorescence intensity). G Western blotting for GLUT1 Serine 226 phosphorylation (n = 6), and ( H ) glucose uptake rate in human RBC treated or not with 1 µM sphingosine for 20 min before a five-minute stimulation or not with 5 nM TPA at 37 °C (n = 5). The ‘n’ in the ( A – H ) refers to samples from individual patients. Data are presented as mean ± sd and tested with stack matched one-way ANOVA followed by Tukey’s ( A – C , G , H ); paired two-tailed t test ( D , F ). p* < 0.05; p** < 0.01; p*** < 0.001; p**** < 0.0001.

Article Snippet: In the case of human recombinant PP2A catalytic subunit (Cayman Chemical, Ann Arbor, USA) 0.58 mU in 100 μl were incubated for 10 min with all substances.

Techniques: Incubation, Activity Assay, Flow Cytometry, Binding Assay, Fluorescence, Western Blot, Phospho-proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells

doi: 10.1038/s41467-023-44109-x

Figure Lengend Snippet: C57Bl6 mice were fed either standard chow diet or high caloric diet (DIO diet) for 12 weeks and the following parameters were determined. A Fasting plasma blood glucose levels (n = 5/6), ( B ) HbA1c (n = 8/8), ( C ) S1P concentrations (n = 11/12), ( D ) PP2A activity (n = 8/7), ( E ) glucose uptake (n = 8 each), and ( F ) Sphk activity (n = 4 each) based on the conversion of C17-sphingosine to C17-S1P as measured by LC-MS/MS (n = 4). G HbA1c levels of human patients with and without T2DM (n = 13 case-controls, characteristics in Supplementary Table ). H Intracellular S1P concentrations (n = 13 each), ( I ) PP2A activity (n = 10/9) and ( J ) cell surface GLUT1 (n = 8/6) on RBC from patients with and without T2DM (randomly selected from above groups). K Correlation between intracellular S1P and PP2A activity in RBC from T2DM patients (n = 10). Data are presented as mean ± sd and tested with two-tailed unpaid t test ( A – I ) and two tailed Pearson correlation coefficient ( J ); p* < 0.05; p**<0.01; p**** < 0.0001.

Article Snippet: In the case of human recombinant PP2A catalytic subunit (Cayman Chemical, Ann Arbor, USA) 0.58 mU in 100 μl were incubated for 10 min with all substances.

Techniques: Clinical Proteomics, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Sphingosine-1-phosphate suppresses GLUT activity through PP2A and counteracts hyperglycemia in diabetic red blood cells

doi: 10.1038/s41467-023-44109-x

Figure Lengend Snippet: A Glucose tolerance test in Mfd2b +/+ and Mfsd2b −/− mice either on a normal chow diet or on a DIO diet for 8 weeks. Inset shows area under the curve (n = 5). B HbA1c (n = 5 each) and ( C ) TBARS levels (n = 4/3) in the same mice as determined by MDA measurements. D 13 C 2 PRPP relative exchange rate in metabolic flux experiments using 1,2,3- 13 C 3 -D-glucose (20 min incubation); n = 6/8 Mfd2b +/+ and 4/4 Mfsd2b –/– . E 13 C 3 lactate/ 13 C 2 lactate ratio in the same experiment (n = 5/9 Mfd2b +/+ and 4/4 Mfsd2b –/– ). F 13 C 3 2,3-BGP relative exchange rate from the same experiment (n = 7/9 Mfd2b +/+ and 4/4 Mfsd2b –/– ). Data are presented as mean ± sd and tested with one-way ANOVA ( A ) and ordinary two-way ANOVA followed by Tukey’s ( B – F ); p* < 0.05; p** < 0.01; p**** < 0.0001. G Schematic of putative S1P functions in RBC: Blood glucose and sphingosine entry regulate Sphk1 activity and S1P production, whereas MFSD2B exports S1P, resulting in dynamic modulation of S1P levels in RBC. S1P associates with the catalytic PP2a subunit and activates the enzyme to reduce GLUT phosphorylation, cell surface localization and glucose uptake. The full arrow indicates dephosphorylation-dependent and the dotted arrow dephosphorylation-independent PP2A-mediated GLUT internalization, respectively. Acute S1P elevation activates glycolysis to compensate for lower glucose entry at the expense of the pentose phosphate pathway (PPP), whereas chronically high S1P RBC levels as in Mfsd2b –/– RBC retain normal glycolysis but increase PPP metabolic fluxes to produce reducing equivalents for protection against hyperglycemia-induced lipid peroxidation.

Article Snippet: In the case of human recombinant PP2A catalytic subunit (Cayman Chemical, Ann Arbor, USA) 0.58 mU in 100 μl were incubated for 10 min with all substances.

Techniques: Incubation, Activity Assay, Phospho-proteomics, De-Phosphorylation Assay