human recombinant pp2a catalytic subunit (cayman chemical, ann arbor, usa)  (Cayman Chemical)

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

Journal: eLife

Article Title: PH domain-mediated autoinhibition and oncogenic activation of Akt

doi: 10.7554/eLife.80148

Figure Lengend Snippet: ( A ) PIP3 binding assays with several isolated Akt PH domain forms. WT: black, R86A: blue, E17K: yellow, Y18A: red, E17K Y18A: green. Fluorescent anisotropy spectra were obtained from the mixture of 50 nM fluorescein-labeled soluble (C8) PIP3 and varying amount of Akt PH domain (0–50 μM). The K D values were determined from fluorescence anisotropy versus PH domain protein concentration plots (n=3, SD shown). ( B ) Full-length Akt mutants have different sensitivities toward the dephosphorylation of pT308 by PP2A phosphatase. Dephosphorylation assays were performed with Akt mutants containing pT308 and lacking C-tail phosphorylation (A6–A9). The dephosphorylation rates were monitored by western blots with anti-pT308 antibody and T 1/2 values shown were determined from plots for fraction of pT308 versus time (n≥3, SEM shown). Figure 5—source data 1. Fluorescence anisotropy binding experiments to measure the phospholipid, phosphatidylinositol 3,4,5-triphosphate (PIP3) binding affinities of the PH domain mutants. Figure 5—source data 2. Dephosphorylation assays using PP2A phosphatase on full-length Akt mutants containing pT308 and a non-phosphorylated C-tail with raw western blot images.

Article Snippet: Chemical compound, drug , PP2A catalytic subunit (human recombinant, L309 deletion) , Cayman Chemical , Cat. No.: 10011237 , .

Techniques: Binding Assay, Isolation, Labeling, Fluorescence, Protein Concentration, De-Phosphorylation Assay, Phospho-proteomics, Western Blot

Journal: eLife

Article Title: PH domain-mediated autoinhibition and oncogenic activation of Akt

doi: 10.7554/eLife.80148

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , PP2A catalytic subunit (human recombinant, L309 deletion) , Cayman Chemical , Cat. No.: 10011237 , .

Techniques: Avidin-Biotin Assay, Recombinant, Software