phosphorylated pp2a  (R&D Systems)

Journal: mBio

Article Title: LincR-PPP2R5C deficiency enhancing the fungicidal activity of neutrophils in pulmonary cryptococcosis is linked to the upregulation of IL-4

doi: 10.1128/mbio.02130-24

Figure Lengend Snippet: IL-4 activated LincR-PPP2R5C KO neutrophils to kill C. neoformans . ( A ) Fungicidal activity of WT and LincR-PPP2R5C KO neutrophils against C. neoformans stimulated with or without IL-4 (15 ng/mL) ( n = 6 per group). ( B ) Western blot (WB) for elastase in WT and LincR-PPP2R5C KO neutrophils stimulated as described in panel A for 4 h. The data are representative of three independent experiments, and the statistical analysis is shown on the right. ( C ) ELISA detection of elastase levels in WT and LincR-PPP2R5C KO neutrophils cocultured with C. neoformans for 4 h under IL-4 stimulation. ( D ) Fungicidal activity of LincR-PPP2R5C KO neutrophils against C. neoformans after treatment with alvelestat or dimethyl sulfoxide. ( E ) MFI of ROS in WT and LincR-PPP2R5C KO neutrophils stimulated as described in panel A for 4 h, as detected by flow cytometry. The statistical diagram is shown on the right. ( F ) WB was used to detect the expression levels of total PP2A, phosphorylated PP2A, and methylated PP2A in WT and LincR-PPP2R5C KO neutrophils after coculture with C. neoformans for 4 h, with or without IL-4 stimulation. The statistical graph is shown on the right. Under the stimulation conditions depicted in panel A , the elastase content ( G ) and the MFI of ROS ( H ) in LincR-PPP2R5C KO neutrophils were detected by ELISA and flow cytometry, respectively, after treatment with okadaic acid (OA, 2 nM) or ethanol. The data are shown as mean ± SEM. * P < 0.05, ** P < 0.01; unpaired Student’s t -test. ns, not significant.

Article Snippet: The blots were then blocked with 5% non-fat dried milk in Tris-buffered saline supplemented with 0.05% Tween 20 (TBST) and incubated with primary antibodies against β-actin (1:2,000, GB15003; Servicebio), total protein phosphatase 2A (PP2A) (1:5,000, ab32104; Abcam), phosphorylated PP2A (1:1,000, sc-271903; Santa Cruz Biotechnology), methylated PP2A (1:1,000, sc-81603; Santa Cruz Biotechnology), and neutrophil elastase (NE) (1:1,000, ab68672; Abcam) overnight.

Techniques: Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Methylation

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: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Soluble epoxide hydrolase-targeting PROTAC activates AMPK and inhibits endoplasmic reticulum stress.

doi: 10.1016/j.biopha.2023.115667

Figure Lengend Snippet: Fig. 4. sEH degradation by ALT-PG2 attenuates thapsigargin-induced ER stress in Huh-7 hepatic cells. Immunoblot analysis of (A) sEH, CHOP, SOCS3 and (B) total and phosphorylated eIF2α in Huh-7 hepatic cells exposed to 10 nM ALT-PG2 for 48 h co-incubated with vehicle or the ER stressor thapsigargin (1 µM) for the last 24 h. (C) TRB3, VLDLR, p65 and TNF-α in Huh-7 hepatic cells exposed to 10 nM ALT-PG2 for 48 h co-incubated with vehicle or the ER stressor thapsigargin (1 µM) for the last 24 h. (D) Immunoblot analysis of CHOP in Huh-7 hepatic cells exposed to 10 nM ALT-PG2 for 48 h co-incubated with vehicle or the ER stressor thap sigargin (1 µM) for the last 24 h with or without the AMPK inhibitor compound C (15 µM). Data are presented as the mean ± SEM. Significant differences were established by one-way ANOVA with Tukey’s post hoc test. *p < 0.05, * *p < 0.01 and * **p < 0.001 vs. CT. #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. thapsi gargin. $$p < 0.01 vs. Thapsi+ALT-PG2.

Article Snippet: Immunoblotting was performed with antibodies against AMPKα (#2532, Cell Signaling Technology, Danvers, MA, USA), phosphorylated AMPK Thr172 (#2531, Cell Signaling Technology), β-Actin (A5441, Sigma), CHOP (GTX112827, Genetex, Irvine, CA, USA), eIF2α (#9722, Cell Signalling Technology), phosphorylated eIF2α Ser51 (#9721, Cell Signalling Technology); ERK1/2 (44/42 MAPK) (#9102, Cell Signaling Technology), phosphorylated ERK1/2 (44/42 MAPK) Thr202/Tyr204 (#9101, Cell Signalling Technology), IRβ (#3025, Cell Signaling Technology), IRS-1 (#2382, Cell Signaling Technology), NF-κB p65 (sc-109, Santa Cruz Biotechnology Inc., Dallas, TX, USA), phosphorylated NF-κB p65 Ser536 (#3036 s, Cell Signalling Technology), PP2A (#2259, Cell Signaling Technology), p53 (2524 T, Cell Signaling Technology), sEH (sc-25797, Santa Cruz Biotechnology Inc.), SOCS3 (sc-9023, Santa Cruz Biotechnology Inc.), STAT3 (sc-482X, Santa Cruz Biotechnology Inc.), phosphorylated STAT3 Tyr705 (#9131, Cell Signaling Technology), TNF-α (AF410-NA, R&D Systems, Minneapolis, MN, USA), TRB3 (sc-365842, Santa Cruz Biotechnology Inc.), vinculin (sc-25336, Santa Cruz Biotechnology Inc.), VLDLR (AF2258, R&D Systems).

Techniques: Western Blot, Incubation