Journal: Journal of Extracellular Biology

Article Title: Effect of a 12‐Week Endurance Training Program on Circulating Extracellular Vesicle Proteome in Sedentary Adults With Obesity

doi: 10.1002/jex2.70087

Figure Lengend Snippet: Extracellular vesicle isolation method from plasma samples. (A) Differential centrifugations followed by filtration and size exclusion chromatography (SEC). (B) Protein concentration (line) on the 14 SEC fractions, combined with western blot analysis using EV‐markers (ALIX and CD9) and Apolipoprotein A1 (APO A1) and B (APO B) as non‐EV markers on fractions 7 to 14. Fractions 7 to 9 are considered EV‐rich and protein‐ and lipoprotein‐poor and were pooled. (C) Transmission electron microscopy image of the pooled fractions (negative staining), scale bar refers to 1 µm and 200 nm. (D) Particles size distribution in pooled fractions analysed by NTA.

Article Snippet: The following antibodies were applied: Apolipoprotein (APO) B (1:1000, Santa Cruz, sc‐376818), APO A1 (1:1000, Santa Cruz, sc‐393636), ALG‐2‐interacting protein X (ALIX) (1:500, Cell Signaling Technology (CST), E6P9B), cluster of differentiation (CD) 9 (1:1000, CST, D3H4P), CD63 (1:1000, Santa Cruz, sc‐5275), CD81 (1:1000, CST, D3N2D), tumour susceptibility gene 101 (TSG101) (1:1000, Abcam, Ab30871), calnexin (1:1000, CST, 2433), LC3b (1:1000, Sigma, SAB4200361), TOM20 (1:1000, CST, 42406), peroxisome proliferator‐activated receptor gamma, coactivator 1 alpha (PGC‐1α) (1:1000, Santa Cruz, sc517380), oxidative phosphorylation (OXPHOS) (1:1000, Abcam, Ab110413 ), vascular endothelial growth factor A (VEGF‐A) (1:500, Santa Cruz, sc‐7269), citrate synthase (CS) (1:1000, CST, D7V8B), succinate dehydrogenase A (SDHA) (1:1000, Santa Cruz, sc‐390381), extracellular signal‐regulated kinase (ERK) 1/2 (1:1000, CST, L34F12), p‐ERK 1/2 (1:1000, CST, D13.14.4E), NFκB (1:1000, CST, D14E12), p‐NFκB (1:1000, CST, 93H1), hypoxia inducible factor 1 alpha (HIF‐1α) (1:500, CST, D2U3T), peroxiredoxin (PRDX) 1 (1:1000, CST, D5G12), actin (1:5000, BD Biosciences, 612656) and eukaryotic translation elongation factor 2 (eEF2) (1:1000, CST, 2332).

Techniques: Isolation, Clinical Proteomics, Filtration, Size-exclusion Chromatography, Protein Concentration, Western Blot, Transmission Assay, Electron Microscopy, Negative Staining

Journal:

Article Title: Definition of the Residues Required for the Interaction between Glycine-extended Gastrin and Transferrin In Vitro

doi: 10.1111/j.1742-4658.2009.07186.x

Figure Lengend Snippet: A. Following injection of apo-transferrin (10μg/ml) into the BIAcore channel an interaction was observed with both Gamide (red line) and Ggly (blue line) by surface plasmon resonance. After removal of apo-transferrin from the running buffer (thick arrow) the interaction between Ggly/Gamide and apo-transferrin gradually declined. B. Upon injection of holo-transferrin (10μg/ml) into the BIAcore channel no interaction was observed with Gamide (red line) or Ggly (blue line). C. The interaction between Ggly and apo-transferrin was also detected using covalent cross-linking. [125I]-Ggly2–17 was pre-reacted with the bivalent crosslinker disuccinimidyl suberate before mixing with apo-transferrin in 50mM Hepes buffer, pH 7.6 in the absence or presence of increasing concentrations of unlabelled Ggly. The apo-transferrin Ggly complex was separated from the unreacted Ggly by SDS polyacrylamide gel electrophoresis, and the extent of incorporation of radioactivity was determined by phosphoimager and densitometric analysis. Unlabelled Ggly inhibited the interaction in a dose-dependent manner. Lack of interaction between Ggly and holo- transferrin was also confirmed. D. The IC50 for binding of Ggly to apo-transferrin was found to be 39 ± 1 μM by curve-fitting, with an intercept of 92.3%. Data points are means ± SEM, where n=3.

Article Snippet: Following injection of apo-transferrin (10μg/ml) into the BIAcore channel an interaction was observed with both Gamide (red line) and Ggly (blue line) by surface plasmon resonance.

Techniques: Injection, SPR Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Methods for Identification of CA125 from Ovarian Cancer Ascites by High Resolution Mass Spectrometry

doi: 10.3390/ijms13089942

Figure Lengend Snippet: ( A ) Measured values in absorbance units for CA125 standards (0–400 kU/L) (blue), 1:20 diluted P95 serum (green) and 1:20 diluted P95 serum spiked with 80 μg fibronectin (red). The values obtained from P95 serum and spiked P95 serum were used for the Mann-Whitney U test; ( B ) Standard curve obtained by plotting the detected absorbance units against the kU/L of the CA125 standards. This curve was used to calculate the CA125 concentration of the used samples (C) CA125 concentration measured by a first generation ELISA (M11-like). Green: P95 serum, red: P95 serum spiked with 80 μg fibronectin. The increase in reading is statistically significant (* p < 0.05). Error bars indicate standard deviation of measured values within the respective sample; ( D ) CA125 concentrations measured by a clinically used second generation ELISA. I: 500 μL P607 serum + 100 μL H 2 O; II: 500 μL P607 serum + 100 μL apo-serotransferrin (100 mg/mL); III: 500 μL P607 serum + 100 μL holo-serotransferrin (100 mg/mL). The increase in the reading for CA125 is statistically significant (*) between apo-serotransferrin (II) and unspiked serum (I) and statistically highly significant (** p < 0.01) between holo-serotransferrin (III) and unspiked serum (I). Error bars indicate the standard deviation of the measured values in the respective sample.

Article Snippet: Apo-serotransferrin (Merck) and holo-serotransferrin (Merck) were resuspended in H 2 O to a concentration of 100 mg/mL and 100 μL were mixed with 500 μL serum of P607.

Techniques: MANN-WHITNEY, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation