Journal: Journal of animal science and biotechnology

Article Title: Prepartum body conditions affect insulin signaling pathways in postpartum adipose tissues in transition dairy cows.

doi: 10.1186/s40104-019-0347-4

Figure Lengend Snippet: Fig. 4 Western blot detection of insulin signaling proteins in subcutaneous adipose tissues. These tissues were collected on day 2 postpartum from CON cows (n = 3) and OVE cows (n = 3). a Panels of INSR, IRS1, p-IRS1, AKT, p-AKT (Thr308) and p-AKT (Ser473) protein. β-actin was measured as an internal control. b Intensities of INSR, IRS1, p-IRS1, AKT, p-AKT (Thr308) and p-AKT (Ser473) bands were determined using Quantity One software. The results are presented as the ratio of INSR band intensity to the β-actin band intensity, the ratio of p-ISR1 band intensity to IRS1 band intensity, and the ratio of p-AKT (Thr308) and p-AKT (Ser473) band intensities to the AKT band intensity. IRS1, insulin receptor substrate 1; AKT, protein kinase B

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-protein kinase B (AKT) (#9272S; 1:1,000 dilution), rabbit monoclonal anti-phospho(p)-AKT (Ser473) (#9272; 1:2,000 dilution), rabbit monoclonal anti-p-AKT (Thr308) (D25E6; 1:1,000 dilution), mouse monoclonal anti-INSR (ab69508; 1:1,000 dilution), rabbit polyclonal anti-insulin receptor substrate 1 (IRS1) (#2382; 1:1,000 dilution), rabbit polyclonal anti-p-IRS1 (#2381; 1:1,000 dilution) (Cell Signaling Technology, Inc., Danvers, MA, USA), and mouse anti-β-actin Mab (66009–1-Ig; 1:1,000 dilution; Proteintech Group Inc., Chicago, IL, USA).

Techniques: Western Blot, Control, Software

Journal: Journal of dairy science

Article Title: MicroRNA-221 regulates proliferation of bovine mammary gland epithelial cells by targeting the STAT5a and IRS1 genes.

doi: 10.3168/jds.2018-15108

Figure Lengend Snippet: Figure 4. Expression of insulin receptor substrate 1 (IRS1) and signal transducer and activator of transcription 5a (STAT5a) at the mRNA and protein levels in bovine mammary epithelial cells transfected with microRNA (miR)-221 mimic or miR-221 inhibitor. (A) The relative ex- pression of IRS1 and STAT5a was significantly decreased after miR-221 mimic transfection and increased after miR-221 inhibitor transfection; (B) Western blot of IRS1 and STAT5a and their negative controls using GAPDH as a reference control; (C) relative expression of IRS1 and STAT5a proteins after miR-221 mimic or miR-221 inhibitor transfections into bovine mammary epithelial cells (BMEC). Data are presented as mean ± SEM (n = 3); *P < 0.05, **P < 0.01, or ***P < 0.001: significant difference between mimic or inhibitor and respective controls (NC).

Article Snippet: 102 No. 1, 2019 After blocking with 5% skim milk powder solution for 1 h at 37°C, the membranes were incubated overnight at 4°C with a murine polyclonal rabbit antibody against IRS1 and STAT5a (Santa Cruz Biotechnology Inc., Dallas, TX), and then incubated with a horseradish peroxidase (HRP)-conjugated secondary goat-anti-rabbit IgG (Zhongshan-Bio, Beijing, China).

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Communications Biology

Article Title: A xanthene derivative, DS20060511, attenuates glucose intolerance by inducing skeletal muscle-specific GLUT4 translocation in mice

doi: 10.1038/s42003-021-02491-6

Figure Lengend Snippet: a , b Phosphorylation of IRβ, IRS1, Akt (Ser473), and AS160 of the triceps surae muscle excised from C57BL/6 mice ( n = 2) treated with DS20060511 (10 mg kg −1 ), insulin (5 U kg −1 ), or saline as vehicle, via inferior vena cava 10 min after the treatment. c Fluorescence immunostaining of cell surface GLUT4 and intracellular actin fibers in L6-GLUT4myc myotubes treated with 30 μM of DS20060511 or 100 nM of insulin. Arrowheads indicate the characteristic ruffled structure of the polymerized actin and actin-associated surface GLUT4. d GLUT4 translocation activity of 30 μM DS20060511 or 100 nM insulin in the presence of the actin polymerization inhibitor, Latrunculin B, at the indicated concentrations. Values shown are means ± SEM, n = 3. ** P < 0.01 vs. 0 nM Latrunculin B by one-way ANOVA followed by Dunnett’s test. e Concentration-dependent insulin-stimulated GLUT4 translocation in L6-GLUT4myc myotubes with or without 30 μM DS20060511 ( n = 3). f Concentration-dependent DS20060511-stimulated 2-DG uptake with 100 nM insulin in isolated muscles from C57BL/6 mice ( n = 3). Values shown are means ± SEM. *** P < 0.001 by one-way ANOVA followed by Tukey’s test. g Blood glucose levels during ITT in STZ-treated C57BL/6 mice ( n = 6–7). Vehicle or indicated dose of DS20060511 was given orally at the same time as 0.1 U kg −1 insulin injection intraperitoneally. Values shown are means ± SEM. * P < 0.05 vs. vehicle by one-way ANOVA followed by Dunnett’s test. c Scale bar in all panels, 5 μm. Uncropped blots for a and b can be found in Supplementary Fig. .

Article Snippet: Rabbit polyclonal antibody against IRS1 (06-248), mouse monoclonal antibody against phosphotyrosine (05-321), rabbit antiserum against AS160 (07-741), and rabbit polyclonal antibody against phospho-AS160 (07-802) were purchased from Merck Millipore.

Techniques: Phospho-proteomics, Saline, Fluorescence, Immunostaining, Translocation Assay, Activity Assay, Concentration Assay, Isolation, Muscles, Injection

Journal: Experimental and Therapeutic Medicine

Article Title: Ursolic acid ameliorates adipose tissue insulin resistance in aged rats via activating the Akt-glucose transporter 4 signaling pathway and inhibiting inflammation

doi: 10.3892/etm.2021.10901

Figure Lengend Snippet: Effect of UA on IRS-1/PI3K/Akt signaling pathway in eWAT of aged rats. (A) Expression of IRS-1, p-IRS-1 (Ser307), p-IRS-1 (Ser307)/IRS-1, Akt, p-Akt (Ser473) and p-Akt (Ser473)/Akt protein levels. (B) PI3K protein levels. Treatment groups were: Young (untreated, n=8), aged (untreated, n=9), treated with 10 mg/kg UA (UA-L, n=8) or 50 mg/kg UA (UA-H, n=9) for 7 weeks. All animals had free access to a standard diet. *P<0.05 compared with aged group. UA-H, high ursolic acid; eWAT, epididymis white adipose tissue; IRS-1, insulin receptor substrate-1; p, phosphorylated; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.

Article Snippet: Membranes were incubated in blocking buffer (5% skimmed milk) for 2 h at room temperature and immunoblotted with the following primary antibodies for 14 h at 4˚C: Rabbit polyclonal anti-IRS-1 antibody (1:1,000; cat. no. TA312859; Origene Technologies, Inc.), rabbit polyclonal anti-phosphorylated(p)-IRS-1 Ser307 antibody (1:1,000; cat. no. TA325573; Origene Technologies, Inc.), rabbit monoclonal anti-Akt antibody (1:1,000; cat. no. ab179463; Abcam), rabbit monoclonal anti-p-Akt Ser473 antibody (1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), rabbit polyclonal PI3K antibody (1:6,000; cat. no. 2225185; EMD Millipore), rat monoclonal GLUT4 antibody (1:1,000; cat. no. 10613; Santa Cruz Biotechnology, Inc.), rabbit polyclonal NF-κB antibody (1:2,000; cat. no. ab16502; Abcam), rabbit polyclonal IL-1β antibody (1:1,000; cat. no. ab9722; Abcam), rat monoclonal IL-6 antibody (1:1,000; cat. no. ab9324; Abcam), and rabbit polyclonal anti-sodium/potassium-transporting ATPase subunit alpha-1 (ATP-1a1) antibody (1:1,000; cat. no. TA326844; Origene Technologies, Inc.).

Techniques: Expressing