Journal: Journal of Molecular Endocrinology

Article Title: Characterising hyperinsulinemia-induced insulin resistance in human skeletal muscle cells

doi: 10.1530/jme-19-0169

Figure Lengend Snippet: Figure 3 (A) Akt phosphorylation (Ser473), (B) GSK-3β phosphorylation (Ser9) of HMDC cultured in CON or PIE media, (C) AS160 phosphorylation (Ser588) of HDMC’s cultured in CON or PIE media before being stimulated without (open bar) or with (doted bar) insulin (100 nM) for 30 min. Data are mean ± s.e.m. from two to five donors as represented by in the graph. *Significant difference between basal and acute insulin stimulation (P < 0.05).

Article Snippet: Primary antibodies used for analysis Downloaded from Bioscientifica.com at 04/15/2025 07:10:53PM via free access were Akt (#9272), phosphor Akt (Ser473) (#4060), GSK-3β (#9315), GSK-3β (Ser9) (#9336), AS160 (Ser588) (#8730), glucose transporter 4 (GLUT4) (#2213) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2118) purchased from Cell Signalling (NEB, Herts, UK).

Techniques: Phospho-proteomics, Cell Culture

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Exercise effects on γ3-AMPK activity, Akt substrate of 160 kDa phosphorylation, and glucose uptake in muscle of normal and insulin-resistant female rats.

doi: 10.1152/japplphysiol.00533.2021

Figure Lengend Snippet: Figure 2. A: phosphorylated (p) AMP-activated protein kinase (AMPK)aThr172/AMPKa in epitrochlearis muscles immediately postexercise (IPEX). IPEX vs. sedentary (SED) within each diet (P < 0.001). HFD, high-fat diet; LFD, low-fat diet. B: phosphorylated Akt substrate of 160 kDa (AS160)Ser704/AS160 in epi- trochlearis muscles at IPEX. IPEX vs. SED within each diet (P < 0.001 for LFD group; P < 0.01 for HFD group). C: phosphorylated acetyl CoA carboxylase (ACC)Ser79/ACC in epitrochlearis muscles at IPEX. IPEX vs. SED within each diet (P < 0.01). Data were analyzed by 2-way analysis of variance. Tukey post hoc analysis was performed to identify significant differences. Values are means ± SD; n = 12 rats per treatment group. The representative blots are aligned with the group identification in the bar graph.

Article Snippet: Anti-phospho Akt Ser473 (pAktSer473; #9271), anti-phospho Akt Thr308 (pAktThr308; #13038), anti-Akt (#4691), anti-phospho AS160 Thr642 (pAS160Thr642; #8881), anti-phospho AS160 Ser588 (pAS160Ser588; #8730), anti-phospho AMPKa Thr172 (pAMPKaThr172; #2531), anti-AMPK-a (AMPKa; #5831), antiacetyl CoA carboxylase (ACC; #3676), anti-phospho ACC Ser79 (pACCSer79; #3661), anti-hexokinase II (HKII; #2867), and anti-rabbit IgG horseradish peroxidase (HRP) conjugate (#7074) were from Cell Signaling Technology (Danvers, MA).

Techniques: Muscles

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Exercise effects on γ3-AMPK activity, Akt substrate of 160 kDa phosphorylation, and glucose uptake in muscle of normal and insulin-resistant female rats.

doi: 10.1152/japplphysiol.00533.2021

Figure Lengend Snippet: Figure 6. A: phosphorylated (p) Akt substrate of 160 kDa (AS160)Ser704/AS160 in paired epitrochlearis muscles incubated ± 100 mU/mL insulin at 3 h post- exercise (3hPEX). HFD, high-fat diet; LFD, low-fat diet; SED, sedentary. 3hPEX vs. SED within each diet with no insulin (P < 0.01 for LFD group; P < 0.05 for HFD group); †3hPEX vs. SED within each diet with insulin (P < 0.05 for LFD group; P < 0.01 for HFD group). B: delta-insulin pAS160Ser704/AS160. C: pAS160Ser588/AS160 in paired epitrochlearis muscles incubated ± 100 mU/mL insulin at 3hPEX. 3hPEX vs. SED within each diet with no insulin (P < 0.01 for LFD group; P < 0.05 for HFD group); †3hPEX vs. SED within LFD group with insulin (P < 0.05). D: delta-insulin pAS160Ser588/AS160. E: pAS160Thr642/ AS160 in paired epitrochlearis muscles incubated ± 100 mU/mL insulin at 3hPEX. †3hPEX vs. SED within each diet with insulin (P < 0.05). F: delta-insulin pAS160Thr642/AS160. Data were analyzed by 2-way analysis of variance within each insulin level (without or with insulin) or for delta-insulin values. Delta- insulin values= value with insulin value without insulin from paired muscles. Tukey post hoc analysis was performed to identify significant differences. Values are means ± SD; n = 10 rats per treatment group. The representative blots are aligned with the group identification in the bar graph.

Article Snippet: Anti-phospho Akt Ser473 (pAktSer473; #9271), anti-phospho Akt Thr308 (pAktThr308; #13038), anti-Akt (#4691), anti-phospho AS160 Thr642 (pAS160Thr642; #8881), anti-phospho AS160 Ser588 (pAS160Ser588; #8730), anti-phospho AMPKa Thr172 (pAMPKaThr172; #2531), anti-AMPK-a (AMPKa; #5831), antiacetyl CoA carboxylase (ACC; #3676), anti-phospho ACC Ser79 (pACCSer79; #3661), anti-hexokinase II (HKII; #2867), and anti-rabbit IgG horseradish peroxidase (HRP) conjugate (#7074) were from Cell Signaling Technology (Danvers, MA).

Techniques: Muscles, Incubation

Journal: Frontiers in Physiology

Article Title: Impact of PCSK9 on CTRP9-Induced Metabolic Effects in Adult Rat Cardiomyocytes

doi: 10.3389/fphys.2021.593862

Figure Lengend Snippet: Impact of PCSK9 on CTPR9-induced effects on glucose metabolism. Adult rat cardiomyocytes were incubated in the presence of LacZ-containing supernatant (control), murine wild-type PCSK9 or GOF PCSK9 (each 0.5 μg/ml) in serum-free medium for 24 h and subsequently treated with CTRP9 (4 μg/ml) for the indicated times. (A) Phosphorylation of Akt and AS160 was analyzed after 20 min CTRP9 stimulation by Western Blotting. Representative Western blots and densitometry of protein data are shown. Total Akt and total AS160 as well as GAPDH served as a loading control. (B) Translocation of GLUT-4 from the cytosol to the plasma membrane was analyzed after 30 min CTRP9 stimulation. Representative Western blots and densitometry of protein data are shown. Na-K-ATPase served as a marker for membrane fraction and GAPDH as a marker for the cytosol. (C) Glucose uptake was measured after 10 min stimulation with CTRP9. (D) Changes in GLUT-4, hexokinase and PFK1 mRNA expression after 24 h stimulation with CTRP9 were analyzed by Real-time PCR. All data are mean ± SEM, 4 independent experiments, n = 8 per group, * p < 0.05.

Article Snippet: Following blocking, filters were incubated with antibodies directed against, phospho-AMPK (Thr172), alpha-AMPK, phospho-Akt (Thr308), total-Akt, phospho-AS160 (Thr642), total-AS160 (all Cell Signaling Technology), PCSK9 (Abcam), ND1 (Abcam), Cox I (Thermo Fisher Scientific), Na-K-ATPase and GAPDH (both Abcam).

Techniques: Incubation, Control, Phospho-proteomics, Western Blot, Translocation Assay, Clinical Proteomics, Membrane, Marker, Expressing, Real-time Polymerase Chain Reaction

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Liver acts as a metabolic gate for the traumatic brain injury pathology: Protective action of thyroid hormone

doi: 10.1016/j.bbadis.2023.166728

Figure Lengend Snippet: List of Antibodies.

Article Snippet: pAS160 , Cell signaling , 8619 , 160.

Techniques: Molecular Weight

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Liver acts as a metabolic gate for the traumatic brain injury pathology: Protective action of thyroid hormone

doi: 10.1016/j.bbadis.2023.166728

Figure Lengend Snippet: Direct effect of T4 on insulin signaling and fat metabolism in primary hepatocytes: Representative western blots and densitometry compared to total/β-actin; a) FAS (one way ANOVA, F (3,20) = 3.09, P = 0.0502); b) pAMPK (thr172) (one way ANOVA, F (3, 19) = 7.05, P = 0.0022); c) pAS160 (one way ANOVA, F (3, 20) = 10.04, P = 0.0003); d) pAKT (ser 473) (one way ANOVA, F (3, 20) = 2.38, P = 0.0996); e) pGSK3β (ser 9) (one way ANOVA, F (3, 20) = 1.22, P = 0.3285); f) Relative 2-NBDG Suorescence (A.U.) (one way ANOVA, F (3, 20) = 22.13, P 0.0001); g) Correlation between levels of pAMPKα (thr172)/AMPK and relative 2-NBDG fluorescence (A.U.). Three independent experiments were performed in replicates. Data are expressed as mean ± SEM. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. Data were analyzed using one-way ANOVA followed by Holm-Sidak post hoc test.

Article Snippet: pAS160 , Cell signaling , 8619 , 160.

Techniques: Western Blot, Fluorescence

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Akt Substrate of 160 kD Regulates Na + ,K + -ATPase Trafficking in Response to Energy Depletion and Renal Ischemia

doi: 10.1681/ASN.2013101040

Figure Lengend Snippet: Energy depletion leads to AS160 dephosphorylation at S588. (A) The apparent molecular weight of AS160 decreases as a result of its dephosphorylation. MDCK cells are subjected (AA/DG) or not (−) to energy depletion for 1 hour. Total cell lysates are incubated in the presence (+) or absence (−) of 10 units of CIP for 30 minutes at 37°C. Lysates are blotted with anti-AS160 antibody to visualize the shift of the apparent molecular weight of AS160. Blots are also probed with anti–phospho-AMPK and p-AS160 S588 antibodies to document the efficacy of both the ATP depletion and the CIP treatments. Anti–β-actin is also used to assess the total amount of protein loaded. (B) Total MDCK cells lysates are blotted with rabbit anti-AS160 to detect the endogenous AS160 and with phospho-specific antibodies directed against the AS160 phosphorylation sites S588 and T642. Lysates are also probed with antibodies directed against p-AMPK. Blots are probed with anti–β-actin to assess the total amount of protein loaded. The data indicate that energy depletion is associated with a decrease in the apparent molecular weight of AS160 that is induced by the dephosphorylation of AS160 at S588. (C) Quantification of AS160 phosphorylation at S588 and T642 is based on four independent experiments. *P<0.01. Typical results of one of four experiments are shown. CIP, calf intestinal alkaline phosphatase; p-AMPK, phospho-AMPK.

Article Snippet: 45 Polyclonal phospho-specific antibodies recognizing AS160 phosphorylated at S588 and T642 were obtained from Symansis (New Zealand).

Techniques: De-Phosphorylation Assay, Molecular Weight, Incubation