Journal: Biochemical Journal

Article Title: Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate

doi: 10.1042/BCJ20220153

Figure Lengend Snippet: ( A ) 3T3-L1 adipocytes stably expressing HA-TRARG1, HA-TRARG1-7A or HA-TRARG1-7E were serum-starved for 2 h. Cells were fixed and stained for nuclei (DAPI, blue ), GLUT4 ( red ) and HA ( green ). Immunofluorescence imaging was performed by confocal microscopy. Instances of colocalization between GLUT4 and HA-TRARG1, HA-TRARG1-7A or HA-TRARG1-7E are indicated by closed arrowheads. Scale bar, 20 µm. ( B ) Immunoblotting analysis of wild-type TRARG1 (T1) or TRARG1 truncation mutants expressed in HEK-293E cells. del_101–127 and del_129–173 mutants were phosphorylated as indicated by the apparent higher molecular weight bands; del_101–173 mutant was not phosphorylated as indicated by the lack of apparent higher molecular weight band. ( C ) N-terminally HA-tagged and C-terminally eGFP fused TRARG1 construct (HA-TRARG1-eGFP) and its truncation mutants were expressed in HEK-293E cells. Subcellular localization of these constructs was determined by confocal microscopy. Full-length TRARG1 and del_101–127 mutant were localized to the PM; del_129–173 mutant was localized to intracellular membranes; del_101–173 mutant was cytosolic. ( D ) Serum-starved or insulin-stimulated 3T3-L1 adipocytes were subjected to subcellular fractionation. Subcellular localization of GSK3 was determined by immunoblotting analysis. Tubulin and caveolin1 were immunoblotted to control for loading of cytosolic and PM proteins, respectively. ( E ) Knockdown efficiency of Trarg1 as assessed by qPCR (**** P < 0.0001, comparison with non-targeting control siRNA). ( F ) 3T3-L1 adipocytes were serum-starved in the absence (DMSO) or presence of GSK3 inhibitors (10 µM CHIR99021; CHIR or 500 nM LY2090314; LY) before treatment with or without 0.5 nM or 100 nM insulin for 20 min. Surface GLUT4 was quantified by immuno-labeling and expressed relative to cell number as measured by nuclei number ( n = 5, mean ± SEM, # P < 0.05 , ## P < 0.01, ### P < 0.001, #### P < 0.0001, compared with the DMSO condition with the same insulin treatment and gene knockdown; ‡ P < 0.05, ‡‡‡‡ P < 0.0001, compared with the non-targeting (NT) knockdown with the same insulin and drug treatment). ( G ) Differences in PM GLUT4 between CHIR- or LY-treated and DMSO control condition with the same insulin treatment and gene knockdown as shown in ( F ) were calculated (mean ± SEM, * P < 0.05, compared with NT knockdown with the same insulin and drug treatment). For panel B and D, the migration positions of molecular mass markers (kilodaltons) are shown to the right. Higher molecular weight (HMW) TRARG1 bands indicated by arrow.

Article Snippet: Cells were blocked and permeabilized with 2% (w/v) BSA and 0.1% (w/v) saponin in PBS followed by incubation with a mixture of rabbit anti-HA antibody (CST, C29F4) and anti-GLUT4 1F8 antibody (generated in-house) at RT for 45 min.

Techniques: Stable Transfection, Expressing, Staining, Immunofluorescence, Imaging, Confocal Microscopy, Western Blot, Molecular Weight, Mutagenesis, Construct, Fractionation, Control, Knockdown, Comparison, Immunolabeling, Migration

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Insulin stimulation fails to increase glucose uptake in human airway epithelia expressing F508del-CFTR. A: primary hBE cells were treated with bilateral insulin (black bars) for a total of 15 min in the presence of 2-deoxy-d-[3H]glucose to measure glucose uptake and retention within the cell layer. Cells were also treated with cytochalasin B (gray bars) to inhibit fusion of Glut4 storage vesicles (GSVs) to the plasma membrane. Unstimulated control cells are represented by white bars. NhBE cells responded to insulin by increasing their glucose uptake, whereas CFhBE cells did not respond to insulin. Cytochalasin B (CytoB) treatment prevented the full effect of insulin stimulation in NhBE cells but had no effect in CFhBE cells. B: comparably treated NuLi-1 and CuFi-5 cells had insulin stimulation responses similar to those seen in NhBE and CFhBE, respectively; n = 4 filters for each data point for each graph. C: temporal glucose uptake measurements indicated that CuFi-5 cells did not respond to treatment with bilateral insulin (■) and behaved similarly to unstimulated CuFi-5 cells (□) and unstimulated NuLi-1 cells (○) compared with stimulated NuLi-1 cells (●). D: NuLi-1 or CuFi-5 filters were pretreated with apical insulin solution for 15 min before measuring the uptake of 2-deoxy-d-[3H]glucose. The basolateral solution contained neither glucose nor insulin. Apical stimulation resulted in apical glucose uptake in NuLi-1 cells but not in CuFi-5 cells; n = 18 filters for each data bar. All data are shown as means ± SE where *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by unprotected two-way ANOVA Fisher’s LSD test.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Expressing, Clinical Proteomics, Membrane, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Glut4 protein is detectable in human tracheal and bronchial epithelial cells by immunohistochemistry. Glut4 appears to be expressed by ciliated cells (black arrows) and by nonciliated cells (white arrows) in the human trachea (A) and bronchioles (C). Blood vessels (arrowheads) are located within 50–100 μm from the tracheal and bronchial epithelial cells. B, D, and F: control sections processed with nonspecific isotype control antibody. E: adipose tissue was used as a positive control tissue where Glut4 is known to be expressed.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Immunohistochemistry, Control, Positive Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Human airway epithelial cells express proteins necessary for insulin-stimulated glucose transport. A: quantitative RT (qRT)-PCR analysis of a subset of glucose transporter and related gene transcripts in freshly obtained human nasal epithelial (hNE) cells and air-liquid interface-cultured primary human bronchial epithelial (hBE) cells. White, freshly isolated hNE; gray, NhBE; hatched gray, cultured NuLi-1 cells; black, CFhBE; hatched black, cultured CuFi-5 cells. HKG, average of three housekeeping genes (ACTB/GAPDH/HPRT1). The official gene symbols and common names of the full qRT-PCR screen are given in Table 1. A cycle threshold (Ct) result of 35 is considered not expressed (broken line). Data are shown as means ± SE; n = 4 freshly isolated nasal epithelial scrapes and n = 3 cultured filters from 3 different donors. B: gel electrophoresis results demonstrate a primer dimer in glucose transporter (Glut) 4 PrimePCR amplification products (left), which may explain the high Ct values observed; an additional band below the expected size was detected in reactions not containing template. The remaining genes detected produced single bands as PrimePCR amplified gene products in the freshly isolated nasal curretage samples (right). C and D: immunoblots demonstrating protein expression of the insulin receptor, β-subunit (IR-β), and insulin-stimulated Glut4 in primary hBE cells (C), T84 colon carcinoma cells, and HeLa cervical adenocarcinoma cells and NuLi-1 and CuFi-5 human airway cells (D). E: immunoblots demonstrating Glut1 expression in NuLi-1 and CuFi-5 human airway cells compared with HEK293 cell lysates containing overexpressed Glut1-FLAG protein. F: immunoblots demonstrating Glut10 expression in NuLi-1 and CuFi-5 human airway cells compared with HEK293 cell lysates containing overexpressed Glut10-FLAG protein.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Quantitative RT-PCR, Cell Culture, Isolation, Nucleic Acid Electrophoresis, Amplification, Produced, Western Blot, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Gene list and qRT-PCR results from freshly isolated nasal epithelia and cultured bronchial epithelia

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Isolation, Cell Culture

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Primary and immortalized human airway epithelia exhibit apical organization of Glut4 storage vesicles. A and B: primary human airway epithelial cells without insulin stimulation show Glut4 (green) lateral to the nuclei (blue) in primary NhBE cells but show only apical organization relative to the nuclei in primary CFhBE cells. Apical β-catenin is pseudocolored in red to indicate cell borders. C and D: NuLi-1 and CuFi-5 cells showed prominent apical localization of Glut4 (green) relative to the nuclei (blue). Images were collected at ×60 magnification and generated with National Institutes of Health ImageJ software.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Generated, Software

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Model for CFTR-mediated regulation of the airway glucose barrier. A: in normal airway epithelia without insulin stimulation, nutrients like glucose (red) slowly traverse the tight junction-regulated paracellular pathway (purple) while constitutive glucose transporters (blue) mediate baseline glucose metabolism and clearance from the paracellular space. Activity of CFTR (green) is normal, whereas Glut4 transporter (yellow) is stored in GSVs near the apical plasma membrane. B: airway epithelia stimulated with insulin decrease the rate of paracellular flux while simultaneously reducing the amount of glucose in the apical space through the activity of translocated Glut4 in the apical plasma membrane. C: in cystic fibrosis airway epithelia, the paracellular route is less stringent (dashed purple box) compared with normal epithelia. This results in more glucose being transported through the tight junction-mediated barrier, where the constitutive glucose transporters act to reduce the amount of glucose in the apical space. CFTR activity is reduced, effectively changing the fluid and ion balance of CF airway epithelia. D: in CF epithelia, insulin fails to activate GSV translocation, effectively reducing insulin-stimulated Glut4 activity while simultaneously allowing small molecules <10 kDa through the paracellular barrier, effectively allowing nutrient accumulation in the apical space.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Activity Assay, Clinical Proteomics, Membrane, Translocation Assay

Journal: Aging (Albany NY)

Article Title: Gastrodin ameliorates cognitive dysfunction in diabetes by inhibiting PAK2 phosphorylation

doi: 10.18632/aging.204970

Figure Lengend Snippet: Gastrodin intervention suppressed diabetes induced PAK2 phosphorylation and activated PI3K/AKT/GLUT4 pathway. Western blot analysis of p-PAK2 ( A ) PI3K ( B ) p-AKT ( C ) and GLUT4 ( D ) protein expression in the hippocampus of the NC9W, DM9W+S, DM9W+G60 and DM9W+G120 groups. Bar graphs represented optical density of these factors normalized with β-actin, while p-PAK2 and p-AKT were further normalized with total-PAK2 and total-AKT respectively. *p < 0.05 and **p < 0.01.

Article Snippet: The following primary antibodies were used for this study: rabbit anti- phosphatidylinositol 3-kinase (PI3K) antibody (1:2000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-AKT antibody (1:2,000 dilution; ABclonal, Woburn, MA, USA), rabbit anti-p-AKT antibody (1:1,000 dilution; CST), rabbit anti-PAK2 antibody (1:2000 dilutions; Abcam), rabbit anti-p-PAK2 antibody (1:1000 dilution; CST), mouse anti-GLUT4 antibody (1:2000 dilution; Santa Cruz, Dallas, TX, USA) and β-tubulin (1:2,000; Santa Cruz).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Gastrodin ameliorates cognitive dysfunction in diabetes by inhibiting PAK2 phosphorylation

doi: 10.18632/aging.204970

Figure Lengend Snippet: Gastrodin intervention restored the expression of PI3K/AKT/GLUT4 pathway in the hippocampal neurons of diabetic rats. Double immunofluorescence staining showed PI3K, p-AKT and GLUT4 positive neurons in the hippocampus of NC9w ( A ) DM9w+S ( B ) DM9w+G60 ( C ) and DM9w+G120 ( D ) groups. Note the diminution of these factors’ immunofluorescence in the neurons of the DM9w+S group as compared with the normal control. However, the immunofluorescence was restored to a level comparable to that of the normal in the DM9w+G60 group. White arrows indicated double positive cells. Bar = 50 μm.

Article Snippet: The following primary antibodies were used for this study: rabbit anti- phosphatidylinositol 3-kinase (PI3K) antibody (1:2000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-AKT antibody (1:2,000 dilution; ABclonal, Woburn, MA, USA), rabbit anti-p-AKT antibody (1:1,000 dilution; CST), rabbit anti-PAK2 antibody (1:2000 dilutions; Abcam), rabbit anti-p-PAK2 antibody (1:1000 dilution; CST), mouse anti-GLUT4 antibody (1:2000 dilution; Santa Cruz, Dallas, TX, USA) and β-tubulin (1:2,000; Santa Cruz).

Techniques: Expressing, Double Immunofluorescence Staining, Immunofluorescence, Control

Journal: Aging (Albany NY)

Article Title: Gastrodin ameliorates cognitive dysfunction in diabetes by inhibiting PAK2 phosphorylation

doi: 10.18632/aging.204970

Figure Lengend Snippet: The effects of Gastrodin intervention and PAK2 inhibition on the PI3K/AKT/GLUT4 pathway in the primary hippocampal neurons during hyperglycemia. ( A ) CCK-8 analysis of hippocampal neurons exposed to 50 mM glucose in the NC, DM, 2 μM, 5 μM, 10 μM and 20 μM of FRAX597 intervention groups. Western blot analysis of PAK2 ( B ), PI3K ( C ), p-AKT ( D ) and GLUT4 ( E ) protein expression in the primary hippocampal neurons of the NC, DM, DM+G and DM+I groups. Bar graphs represented optical density of these factors normalized with β-actin. *p < 0.05, **p < 0.01 and ***p < 0.001.

Article Snippet: The following primary antibodies were used for this study: rabbit anti- phosphatidylinositol 3-kinase (PI3K) antibody (1:2000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-AKT antibody (1:2,000 dilution; ABclonal, Woburn, MA, USA), rabbit anti-p-AKT antibody (1:1,000 dilution; CST), rabbit anti-PAK2 antibody (1:2000 dilutions; Abcam), rabbit anti-p-PAK2 antibody (1:1000 dilution; CST), mouse anti-GLUT4 antibody (1:2000 dilution; Santa Cruz, Dallas, TX, USA) and β-tubulin (1:2,000; Santa Cruz).

Techniques: Inhibition, CCK-8 Assay, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Gastrodin ameliorates cognitive dysfunction in diabetes by inhibiting PAK2 phosphorylation

doi: 10.18632/aging.204970

Figure Lengend Snippet: Gastrodin intervention and PAK2 inhibition restored the expression of PI3K/AKT/GLUT4 pathway in the primary hippocampal neurons exposed to high glucose. Double immunofluorescence staining of PI3K, p-AKT and GLUT4with NeuN in the primary hippocampal neurons of NC ( A ), DM ( B ), DM+G ( C ) and DM+I ( D ) groups. Bar = 50 μm.

Article Snippet: The following primary antibodies were used for this study: rabbit anti- phosphatidylinositol 3-kinase (PI3K) antibody (1:2000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-AKT antibody (1:2,000 dilution; ABclonal, Woburn, MA, USA), rabbit anti-p-AKT antibody (1:1,000 dilution; CST), rabbit anti-PAK2 antibody (1:2000 dilutions; Abcam), rabbit anti-p-PAK2 antibody (1:1000 dilution; CST), mouse anti-GLUT4 antibody (1:2000 dilution; Santa Cruz, Dallas, TX, USA) and β-tubulin (1:2,000; Santa Cruz).

Techniques: Inhibition, Expressing, Double Immunofluorescence Staining

Journal: Aging (Albany NY)

Article Title: Gastrodin ameliorates cognitive dysfunction in diabetes by inhibiting PAK2 phosphorylation

doi: 10.18632/aging.204970

Figure Lengend Snippet: Diagram illustrating the effects of diabetes on hippocampal neurons. Diabetes could enhance the phosphorylation of PAK2, which reduces the expression of PI3K and thus the phosphorylation of AKT. It leads to decreased expression of GLUT4, which is important for glucose uptake.

Article Snippet: The following primary antibodies were used for this study: rabbit anti- phosphatidylinositol 3-kinase (PI3K) antibody (1:2000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-AKT antibody (1:2,000 dilution; ABclonal, Woburn, MA, USA), rabbit anti-p-AKT antibody (1:1,000 dilution; CST), rabbit anti-PAK2 antibody (1:2000 dilutions; Abcam), rabbit anti-p-PAK2 antibody (1:1000 dilution; CST), mouse anti-GLUT4 antibody (1:2000 dilution; Santa Cruz, Dallas, TX, USA) and β-tubulin (1:2,000; Santa Cruz).

Techniques: Phospho-proteomics, Expressing