Journal: Journal of Biological Chemistry

Article Title: Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

doi: 10.1074/jbc.m109.012583

Figure Lengend Snippet: FIGURE 4. TP activation impairs insulin signaling in a PTEN-dependent manner. A and B, HUVECs were treated with SQ29548 IBOP and harvested for the analysis of (A) PTEN-Ser380/The382/383 phosphorylation, PTEN levels, and (B) PTEN activity (n 4; *, p 0.05 versus control (Ctrl); †, p 0.05 versus IBOP). C, PTEN is required for IBOP-induced dephosphorylation of Akt and eNOS. HUVECs were transfected for 48 h with either control siRNA or PTEN siRNA (100 nmol/liter) and treated with IBOP (n 3; *, p 0.05 versus control; †, p 0.05 versus control siRNA IBOP). D, PTEN is required for IBOP-induced inhibition of insulin signaling. The siRNA- transfected HUVECs were treated with IBOP and then stimulated with insulin (n 3; *, p 0.05 versus control siRNA insulin; †, p 0.05 versus control siRNA IBOP insulin).

Article Snippet: Recombinant human active PTEN was from R & D Systems, Inc. (Minneapolis, MN).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Control, De-Phosphorylation Assay, Transfection, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

doi: 10.1074/jbc.m109.012583

Figure Lengend Snippet: FIGURE 6. Rho/ROCK participates in TP-induced inhibition of insulin signaling. A, IBOP activates Rho in HUVECs. The cells were preincubated with 4 mol/liter SQ29548 for 20 min, treated with IBOP for 1 h, and lysed for the analysis of active Rho (GTP bound) using Rhotekin pulldown assays. B, ROCK inhibition with Y27632 blocks IBOP-induced dephosphorylation of Akt and eNOS. C, Y27632 abolishes TP-induced increases in total and phosphorylated PTEN. D, Y27632 inhibits IBOP-stimulated PTEN activity. The cells were pretreated with Y27632 (10 mol/liter, 20 min) and then treated with IBOP overnight (n 4; *, p 0.05 versus control; †, p 0.05versusIBOP).E,Y27632blocksIBOP-inducedimpairmentsininsulinresponses.Theblotisrepresentativeof three blots obtained from three independent experiments.

Article Snippet: Recombinant human active PTEN was from R & D Systems, Inc. (Minneapolis, MN).

Techniques: Inhibition, De-Phosphorylation Assay, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

doi: 10.1074/jbc.m109.012583

Figure Lengend Snippet: FIGURE 7. ROCK is an upstream kinase of LKB1, which up-regulates PTEN phosphorylation. A, activity and phosphorylation (at Ser473 and Thr308) of recombinant Akt1 in the presence or absence of recombinant ROCK1 or ROCK2. Akt1 activity was assessed by phosphorylation of glycogen synthase kinase-3 fusion protein. Rep- resentative Western blots from five separate experiments are shown. B, in vitro kinase assays with recombinant LKB1 in the presence or absence of recombinant ROCK1 or ROCK2. Representative Western blots from four separate experiments are shown. C, in vitro kinase assay showing LKB1-mediated PTEN-Ser380/Thr382/383 phos- phorylation in the presence or absence of recombinant ROCK1 or ROCK2. Similar results were obtained in three independent assays. D, Y27632 inhibits IBOP-induced LKB1-Ser428 phosphorylation. The cells were pretreated with Y27632 (10 mol/liter, 20 min) and then exposed to IBOP overnight (n 4; *, p 0.05 versus control; †, p 0.05 versus IBOP).

Article Snippet: Recombinant human active PTEN was from R & D Systems, Inc. (Minneapolis, MN).

Techniques: Phospho-proteomics, Activity Assay, Recombinant, Western Blot, In Vitro, Kinase Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

doi: 10.1074/jbc.m109.012583

Figure Lengend Snippet: FIGURE 9. TP mediates HFD-induced inhibition of both Akt and eNOS in murine aortas. Male C57BL/6J mice were fed a normal diet (ND) or a HFD for 13 weeks and administrated vehicle or SQ29548 (5 g/kg/day) for the following 5 weeks. A–C, aortic homogenates were analyzed for levels of total and phosphorylated eNOS (phosphorylation at Ser1177) (A), Akt (phosphorylation at Ser473) (B), and PTEN (phosphorylation at Ser380/ Thr382/383) (C). D, proposed pathway underlying TP-induced attenuation of insulin signaling.

Article Snippet: Recombinant human active PTEN was from R & D Systems, Inc. (Minneapolis, MN).

Techniques: Inhibition, Phospho-proteomics

Journal: Neural Regeneration Research

Article Title: MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

doi: 10.4103/NRR.NRR-D-23-01064

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in western blot analysis

Article Snippet: PSN1 rabbit polyclonal , 1:1000 , 16163-1-AP , Proteintech, Rosemont, IL, USA.

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

doi: 10.4103/NRR.NRR-D-23-01064

Figure Lengend Snippet: Modulation of AD proteins by miR-502-3p. (A) Immunoblot analysis of amyloid precursor protein (APP), tubulin associated unit (Tau), presenilin-1 (PSN1) and presenilin-2 (PSN2) and -actin protein in hippocampal neurons (HT22) cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. The levels of AD proteins were changed in miR-502-3p agomiR and antagomiR transfected cells. (B) Densitometry analysis of APP, Tau, PSN1 and PSN2 blot shows increased protein levels in miR-502-3p agomiR transfected cells compared with scramble control, while the cells treated with miR-502-3p antagomiR shows a reduced level of all AD proteins compared with agomiRs. For statistical analysis, Student’s t -test was used for analyzing the data between three groups of samples such as scramble control, agomiRs, and antagomiRs. Experiments were performed in triplicates. The P -values < 0.05 were considered statistically significant. AD: Alzheimer’s disease; miR: microRNA.

Article Snippet: PSN1 rabbit polyclonal , 1:1000 , 16163-1-AP , Proteintech, Rosemont, IL, USA.

Techniques: Western Blot, Transfection, Control