Journal: Pharmaceutics

Article Title: New Intracellular Peptide Derived from Hemoglobin Alpha Chain Induces Glucose Uptake and Reduces Blood Glycemia

doi: 10.3390/pharmaceutics13122175

Figure Lengend Snippet: C2C12 cell signaling assays by Western blotting. C2C12 cells previously differentiated into myotubes were treated in the presence or absence of insulin (100 nM), or the indicated intracellular peptide (Ric1, Ric2, Ric3, or Ric4; 100 µM). Induced phosphorylation of Erk ( A ) or Akt ( B ), respectively, pAkt or pErk, were analyzed by Western blotting as described in . Imaging and band intensity measurements were performed using the ImageJ 1.49 software, and quantifications were performed evaluating the relative levels of pErk or pAkt over total Erk or Akt, respectively. The effect of the indicated peptide on the relative phosphorylation levels were expressed using arbitrary density units ( A , B , lower panels). Data are representative of three independent experiments that produced similar results. The statistical comparisons were performed using Student’s t -test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software * p < 0.05; ** p < 0.001.

Article Snippet: Western blot bands were visualized with Super-Signal West Pico Chemiluminescent substrate (Thermo Scientific) using the ChemiDoc™ MP Imaging System (BioRad, Hercules, CA, USA), and quantified using ImageJ 1.49 software.

Techniques: Western Blot, Imaging, Software, Produced

Journal: Pharmaceutics

Article Title: New Intracellular Peptide Derived from Hemoglobin Alpha Chain Induces Glucose Uptake and Reduces Blood Glycemia

doi: 10.3390/pharmaceutics13122175

Figure Lengend Snippet: Glucose uptake and GLUT4 translocation induced by Ric4 in C2C12 cells. C2C12 cells were previously incubated in serum-free and glucose-free DMEM medium, then incubated with HEPES buffer for 30 min and then incubated for a further 30 min in glucose-uptake buffer containing vehicle, Ric4 (100 µM), or insulin (100 nM) in the presence of 3 H-glucose (1 µCi/mL) or 2-NBDG (80 µM). After incubations cells were lysed with 50 μL of 0.1 N NaOH and fluorescence or radiation of aliquots from the lysate were measured ( A ). Epididymal adipose tissue explants (20–25 mg) were incubated in Krebs–Ringer bicarbonate buffer containing glucose 5.5 mM and 1 μCi/mL of 3 H-deoxyglucose supplemented with 2% fatty acid-free for 30 min at 37 °C in the presence or absence of insulin (100 nM) or Ric4 (100 µM). The explants were processed to evaluate the uptake of 3 H-deoxyglucose ( B ). Myotubes were previously incubated in serum-free and glucose-free DMEM medium and then treated with control vehicle PBS or Ric4 (100 µM) for 30 min. Proteins from subcellular fractions: N/ER ( C ), microsomal ( D ), plasma membrane ( E ), were isolated and the expression Glut4 was analyzed by Western blot. Images were quantified using ImageJ 1.49 software. The statistical comparisons were performed using analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software * p < 0.05; # p < 0.05; **** p < 0.0001.

Article Snippet: Western blot bands were visualized with Super-Signal West Pico Chemiluminescent substrate (Thermo Scientific) using the ChemiDoc™ MP Imaging System (BioRad, Hercules, CA, USA), and quantified using ImageJ 1.49 software.

Techniques: Translocation Assay, Incubation, Fluorescence, Isolation, Expressing, Western Blot, Software

Journal: Pharmaceutics

Article Title: New Intracellular Peptide Derived from Hemoglobin Alpha Chain Induces Glucose Uptake and Reduces Blood Glycemia

doi: 10.3390/pharmaceutics13122175

Figure Lengend Snippet: Western blotting for ERK and AKT in epididymal adipose and tibial muscle tissues following ip administration of Ric4 to WT mice. Mice were treated ip with vehicle (Control) or Ric4 (600 µg/kg). Tissues were collected 30 min after the Ric4 administration and processed for Western blots as detailed in . The phosphorylation of either ERK or AKT in epididymal adipose tissue ( A , B ) and tibial muscle tissue ( C , D ) were analyzed using antibodies anti-pErk ( A , C ) or anti-pAkt ( B , D ). Anti-total ERK, anti-total AKT, and β-actin were used for normalizing protein concentration. Imaging and band intensity measurements were performed using ImageJ 1.49 software. Data are representative of three independent experiments that produced similar results. The statistical comparisons were performed using Student’s t -test using GraphPad Prism software * p < 0.05.

Article Snippet: Western blot bands were visualized with Super-Signal West Pico Chemiluminescent substrate (Thermo Scientific) using the ChemiDoc™ MP Imaging System (BioRad, Hercules, CA, USA), and quantified using ImageJ 1.49 software.

Techniques: Western Blot, Protein Concentration, Imaging, Software, Produced