Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Apolipoprotein A-I Increases Insulin Secretion and Production From Pancreatic β-Cells via a G-Protein-cAMP-PKA-FoxO1–Dependent Mechanism

doi: 10.1161/atvbaha.114.304131

Figure Lengend Snippet: Figure 2. Apolipoprotein A-I (ApoA-I) induces cocalization of ATP-binding cassette transporter A1 (ABCA1) and a heterotrimeric Gαs subunit. A, Ins-1E cells were transfected with Gαs siRNA or scrambled siRNA, then lysed and subjected to Western blotting for Gαs and β-actin. B, Quanti- fication of Gαs expression in siRNA-transfected cells. C, Transfected Ins-1E cells were incubated for 1 hour without (white bars) or with (black bars) apoA-I (final concentration, 1 mg/mL) under basal (2.8 mmol/L) glucose conditions. Insulin levels in the medium were quantified by RIA. D, Ins-1E cells were seeded on coverslips, transfected with a vec- tor encoding for HA-Gαs, serum starved for 6 hours, then incubated for 5 minutes with or without apoA-I (final concentration, 1 mg/mL). The cells were fixed, immunostained with a DyLight-488–labeled ABCA1 antibody and an Alexa-657–labeled HA-tag anti- body, and imaged using a total internal reflection fluorescence microscope. Representative images from 3 separate experiments are shown. Arrows indicate points of colocalization. Scale bar, 20 μm. E, Quantification of the Pearson coefficient. All values represent the mean±SD (n=4 for B and C, n=20 fields of view for E). ****P<0.001 compared with controls.

Article Snippet: The cells were fixed in 10% (v/v) neutral buffered formalin (HT501128, Sigma-Aldrich) for 10 min, permeabilized with PBS containing 0.3% (v/v) Triton X-100 and incubated overnight at 4 °C with a DyLight 488-conjugated rabbit anti-mouse ABCA1 antibody (1:250 dilution, NB400-105G, Novus Biologicals, Littleton, CO) and Alexa647-conjugated mouse anti-HA Tag antibody (1:100 dilution, 3444, Cell Signaling, Technology, Beverly, MA).

Techniques: Binding Assay, Transfection, Western Blot, Expressing, Incubation, Concentration Assay, Labeling, Fluorescence, Microscopy