Journal:

Article Title: Isolation and Characterization of Two Proteins from Moraxella catarrhalis That Bear a Common Epitope

doi:

Figure Lengend Snippet: Interaction of proteins with fibronectin and vitronectin determined by dot blot. (A) Binding of vitronectin to UspA1 and UspA2. For this blot, the purified proteins were spotted onto the membrane, blocked, and incubated with human plasma vitronectin. The bound vitronectin was detected with a rabbit antivitronectin conjugate. (B) Binding of fibronectin to UspA1. Purified fibronectin was spotted onto the membrane, blocked, and incubated with purified UspA1. The bound UspA1 was then detected with MAb 17C7. (C) Binding of fibronectin to UspA2. This procedure was done as described for panel B except with UspA2. The amount of protein applied at each spot is indicated on the left.

Article Snippet: After the proteins had been absorbed into the membrane, it was blocked with BLOTTO for 1 h and incubated with human plasma vitronectin (1 μg/ml in BLOTTO; GIBCO BRL, Grand Island, N.Y.) for 4 h at room temperature.

Techniques: Dot Blot, Binding Assay, Purification, Incubation

Journal: Diabetologia

Article Title: Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α

doi: 10.1007/s00125-013-2950-9

Figure Lengend Snippet: miR-221 targets the 3′ UTRs of ADIPOR1 and ETS1 and decreases their protein levels. ( a,b ) TargetScan conserved predicted miR-221 binding site on the 3′ UTRs of ADIPOR1 and ETS1 . ( c,d ) Quantification of dual-luciferase assay in HEK 293 cells co-transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars) and ADIPOR1/2 ( c ) or ETS1 ( d ) 3′ UTR reporter plasmids. ( d ) Two overlapping fragments of the ETS1 3′-UTR were subcloned into separate vectors, indicated (A) and (B). Firefly/renilla luciferase ratio (luc ratio) was normalised to no-UTR controls. Error bars indicate SD ( n = 5). * p < 0.05 (two-tailed Mann–Whitney U test). ( e ) Quantification of QRT-PCR for ETS1 mRNA in human pre-adipocytes transfected with miR-221 mimic (black bars) or control oligonucleotide (white bars). Error bars indicate SD ( n = 3). ( f ) Immunoblots for ADIPOR1, ETS1 and β-actin in human pre-adipocytes transfected with miR-221 mimic (+) or control oligonucleotide. ( g ) Quantification of the immunoblots shown in ( f ). Background-subtracted mean signal ( n = 2) for ADIPOR1 and ETS1, normalised to the loading control (β-actin). Black bars, miR-221 mimic; white bars, control oligonucleotide

Article Snippet: For 3′ untranslated (UTR) reporter assays, JetPrime (Polyplus-transfection SA, Illkirch, France) was used to co-transfect HEK 293 cells (ATCC) with miRNA 3′ UTR target expression vectors for human ETS1 , ADIPOR1 , ADIPOR2 or control (Genecopoeia, Germantown, MD, USA), and human miR-221 mimic (Dharmacon, Lafayette, CO, USA) or scrambled control oligonucleotide, in 24-well plates.

Techniques: Binding Assay, Luciferase, Transfection, Control, Two Tailed Test, MANN-WHITNEY, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand

doi: 10.1371/journal.pone.0161157

Figure Lengend Snippet: (A) Pooled luciferase results from 4 separate experiments (normalized to the mean of empty vector in each experiment). U2OS cells were transfected with ligand (DLL1, DNER, or EV), and separately a population of U2OS cells was transfected to express Notch, the control luciferase Renilla , and TP1, a promoter that expresses firefly luciferase when Notch is activated. The two populations were co-cultured 24 hours after transfection ( trans configuration), and activity read after an additional 24–48 hours of incubation. (B) C2C12 cells (myoblasts) were incubated with differentiation media (2% horse serum) that either had pre-clustered DLL1-fc (1:1), pre-clustered DNER-fc (1:1), un-clustered DNER-fc, or fc only, all at a ratio of 1:150 in media. Cells were incubated for 72 hours, then fixed, and stained for the presence of myosin heavy chain (MHC) and nuclei. By measuring the percent of total nuclei that were inside of differentiated MHC positive myotubes, fusion indexes were calculated. (C) DNER (top left, green) transfected U2OS cells were not labeled by pre-clustered Notch-fc (top middle, red) but DLL1 (bottom left, green) transfected U2OS cells were labeled by pre-clustered Notch-fc (bottom middle, red). Merged images are shown at far right. Scale 10 μM. **** = p value <0.0001. *** = p value 0.002. ns = not significant. DLL1 = Delta-like 1, a known Notch Ligand, DNER = Delta/Notch-like epidermal growth factor (EGF) related receptor, GSI = γ-secretase inhibitor, fc only = rabbit anti-human-fc.

Article Snippet: Transient transfection of cultured cells was accomplished with the Fugene/Optimem (Promega) system and according to the manufacturer’s instructions with the following plasmids at a concentration, unless otherwise stated, of 0.1 μg/well of DNA per plasmid of a 96-well plate: DNER (generous gift of Drs. De Graaff and Sillevis-Smitt, Erasmus Medical Center, Rotterdam the Netherlands, originally from M. Kengaku), DLL1 (EX-Y3540-M11, GeneCopoeia, Rockville, MD, USA), empty vector on a p-receiver backbone (GeneCopoeia, Rockville, MD, USA), Renilla (luciferase control, 0.01 μg/well, pRL-TK, Promega E2241), TP1 (very sensitive intracellular Notch reporter with twelve CSL binding sites converting intracellular Notch activity to firefly luminescence, which is capable of detecting slight perturbations in signaling strength with little background), and full length human Notch1 on a MigR1 backbone (both generous gifts of Dr. Jon Aster).

Techniques: Luciferase, Plasmid Preparation, Transfection, Control, Cell Culture, Activity Assay, Incubation, Staining, Labeling