Journal: Translational Oncology

Article Title: Uncovering Differently Expressed Markers and Heterogeneity on Human Pancreatic Cancer

doi: 10.1016/j.tranon.2020.100749

Figure Lengend Snippet: Blind SELEX to identify potential biomarkers on pancreatic cancer. (A) The schematic workflow of blind SELEX is depicted. After untargeted SELEX to identify differently expressed markers on plasma membrane of PANC-1 cells, aptamers are enriched in vitro . Target ligands were retrieved via affinity pull-down assays using biotinylated aptamers. After affinity pull-down assays, MS/MS is used to achieve peptide fingerprinting. Finally, the binding of aptamer with the target ligand is validated by surface plasmon resonance. (B) Competition assay was performed to determine whether P19 and P1 bind to the same epitope. The fluorescence intensity was quantified using confocal microscopy. *One-way ANOVA test; * P ≤ .05, ** P ≤ .01. (C) Ten percent of polyacrylamide gel electrophoresis was used to separate immobilized protein samples after pull-down with biotinylated P19, P1, and negative control RNAs. Coomassie-stained gels: M (marker), P19 (lane 1), P1 (lane 2), and NC (irrelevant RNA, lane 3). Arrow indicates target antigens. (D) MS/MS spectra of aptamer binding ligand. Peptide matching and MS/MS spectra of P19 (left) and P1 (right) affinity-purified peptides. Inset: amino acid sequence of the parent peptide showing b- and y-ion series coverage. Target epitopes are highlighted in yellow. (E) Biosensor analysis of the mortalin-aptamer interaction. Binding of mortalin to the P19 (left) and P1 (right) was shown. Biotinylated P19 or P1 was bound to a streptavidin-coupled carboxyl methyl dextran surface, and binding was measured using the SPR technique. The increased response unit was shown in a dose-dependent manner.

Article Snippet: Recombinant mortalin was purchased from Novus Biologicals (NBC1-18380).

Techniques: Clinical Proteomics, Membrane, In Vitro, Tandem Mass Spectroscopy, Binding Assay, SPR Assay, Competitive Binding Assay, Fluorescence, Confocal Microscopy, Polyacrylamide Gel Electrophoresis, Negative Control, Staining, Marker, Affinity Purification, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Nogo-B Receptor Directs Mitochondria-Associated Membranes to Regulate Vascular Smooth Muscle Cell Proliferation

doi: 10.3390/ijms20092319

Figure Lengend Snippet: NgBR KD cells show disrupted ER-mitochondria interaction. shControl- and shNgBR-transfected cells were harvested at 48 h post transfection. MAM formation was analyzed by evaluating the expression of MAM-related proteins in ( A ) total cell lysates (FACL-4 and IP 3 R3) and ( B ) crude mitochondrial extracts (FACL-4 and VDAC1); β-actin and COX IV were utilized as the loading controls, respectively. n = 3. ( C ) Cells were incubated with plasmid expressing mCherry fused with a targeting sequence of subunit IV of COX prior to shRNA transfection. PDI was used as an ER marker. Nuclei were stained with Hoechst (blue). Pearson’s correlation coefficient was calculated to measure colocalization of ER and mitochondria on mCherry positive cells. n > 60 cells/group. Scale bar = 10 μm. ( D ) Transmission electron microscopy of negative control (NC; left) and NgBR knockdown KD (right) VSMCs. Arrowheads indicate the MAMs. The minimum ER-mitochondrial distance was quantified. n > 80 ER-mitochondria contact points/group. Scale bar = 500 nm. * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against proliferating cell nuclear antigen (PCNA; BM0104), VDAC1 (PB0478), GRP75 (PB0668), and IP 3 R1 (PB0223) were obtained from Boster (Pleasanton, CA, USA).

Techniques: Transfection, Expressing, Incubation, Plasmid Preparation, Sequencing, shRNA, Marker, Staining, Transmission Assay, Electron Microscopy, Negative Control, Knockdown