Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Toll-like receptor 2 mediates high-fat diet-induced impairment of vasodilator actions of insulin

doi: 10.1152/ajpendo.00578.2012

Figure Lengend Snippet: Saturated FA-stimulated proinflammatory signaling in primary vascular endothelial cells is impaired by siRNA knockdown of TLR2. HAEC were transiently transfected with scrambled control siRNA or siRNA specifically targeting TLR2. Next, cells were incubated for 48 h and serum-starved for 2 h. Cells were then incubated with either BSA or palmitate (200 μM, 1 h). Cell lysates were subjected to immunoblotting with indicated antibodies. A and B: treatment with palmitate stimulated phosphorylation of JNK and NF-κB. C and D: the quantification of 3 independent experiments is shown in bar graphs (means ± SE). E: cells were plated in chamber slides and transfected with siRNA as above. Cells were then incubated with either BSA or palmitate (200 μM, 4 h) and subjected to immunofluorescence staining with anti-NF-κB p65 and anti-rabbit antibody (right). Overlapped image of anti-NF-κB p65 and nucleus staining with Hochest is shown (left). Fluorescence was visualized by Zeiss epifluorescent microscope. *** P < 0.001.

Article Snippet: The image was visualized with a Zeiss inverted epifluorescent microscope (Axio Observer A1).

Techniques: Transfection, Incubation, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology

doi: 10.1128/JVI.00660-18

Figure Lengend Snippet: C terminus of GP3 is translocated into the lumen of the ER. (A) Primary structure of GP3 with signal peptide (SP), hydrophobic region (HR), six conserved cysteines (lines), and glycosylation sites (branches; numbering of sites is for PRRSV-2 strains). The position of the first site differs between PRRSV-1 and PRRSV-2 strains. Glycosylation site 138 is an additional site present in GP3 from the IAF-Klop strain, and GP3 from XH-GD lacks the site at position 152. The graph shows the percent conservation (y axis) of amino acids at each position (x axis) of a consensus sequence compiled from all PRRSV-1 and PRRSV-2 GP3 sequences present in the database. (B) CHO-K1 cells expressing GP3-YFP from 5 different PRRSV strains and (as a control) a type I transmembrane protein (GP4-YFP from equine arteritis virus [EAV]) were treated with digitonin for 1 min and with proteinase K for 66 min. After each time point, the same microscopic field was recorded with an epifluorescence microscope. (C and D) Two additional glycosylation sites were inserted into the C-terminal part of GP3 from VR-2332 (C) or LV (D), and constructs were expressed in BHK cells. Twenty-four h after transfection, cells were lysed and samples subjected to SDS-PAGE and Western blotting with anti-HA antibody. The C-terminal sequences of GP3 (amino acids 227 to 254 in VR-2332 and 228 to 265 in LV) are shown above the blot. Boldface letters indicate the N-glycosylation site inserted by exchange of one amino acid (underlined). The SDS-PAGE mobility of molecular weight makers are indicated at the left side of the blot. Mock, untransfected cells.

Article Snippet: Pictures were recorded using a Zeiss Axio Vert A1 inverse epifluorescence microscope.

Techniques: Sequencing, Expressing, Microscopy, Construct, Transfection, SDS Page, Western Blot, Molecular Weight

Journal: Journal of Virology

Article Title: Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology

doi: 10.1128/JVI.00660-18

Figure Lengend Snippet: Hydrophobic region of GP3 attaches GFP to membranes. (A) Scheme of GFP constructs used in these experiments. GFP-HR3 contains the whole C-terminal part of GP3 from VR-2332 (amino acids 178 to 254). GFP-HR2 contains the C-terminal part of GP3 except for amino acids 230 to 254, and GFP-HR1 contains only the hydrophobic region (amino acids 181 to 200). Localization of GFP and GFP chimeras in transfected CHO cells was visualized with an epifluorescence microscope. (B) CHO cells were transfected with GFP wild type and GFP chimeras, and 24 h after transfection, membrane (Me) and cytosolic (Cyt) fractions were separated by ultracentrifugation and subjected to Western blotting with antibodies against GFP. The SDS-PAGE mobilities of molecular weight makers are indicated at the left side of the blot. Mock, untransfected cells.

Article Snippet: Pictures were recorded using a Zeiss Axio Vert A1 inverse epifluorescence microscope.

Techniques: Construct, Transfection, Microscopy, Western Blot, SDS Page, Molecular Weight