Secondary structure evolution, as a function of time, for the LOX-1 region (140–165) including strands 0 (red bar with middle point around 150) and 1 (red bar with middle point around 156)

<p><b>Copyright information:</b></p><p>Taken from "Molecular dynamics simulation of human LOX-1 provides an explanation for the lack of OxLDL binding to the Trp150Ala mutant"</p><p>http://www.biomedcentral.com/1472-6807/7/73</p><p>BMC Structural Biology 2007;7():73-73.</p><p>Published online 7 Nov 2007</p><p>PMCID:PMC2194713.</p><p></p> Colour code identifying the secondary structure is shown in the figure

Dynamic cross-correlation maps calculated for the wild-type and the mutant LOX-1 proteins

<p><b>Copyright information:</b></p><p>Taken from "Molecular dynamics simulation of human LOX-1 provides an explanation for the lack of OxLDL binding to the Trp150Ala mutant"</p><p>http://www.biomedcentral.com/1472-6807/7/73</p><p>BMC Structural Biology 2007;7():73-73.</p><p>Published online 7 Nov 2007</p><p>PMCID:PMC2194713.</p><p></p> Panels A and C reports the intra-subunit motion correlations in the wild-type, while panels B and D the intra-subunit motion correlations in the mutant. The black and grey squares represent the Cmotion correlations wit

Immunoblotting of EA cells conditioned media probed with anti MMP-1 polyclonal mouse antibodies (upper panel), and TIMP-1 polyclonal mouse antibodies (lower panel).

<p>Optical density analysis was used to semi-quantify the protein secretion levels upon ox-LDL cell-treatment.</p

Western blot of sLOX-1 released from cells treated with MβCD and different protease inhibitors.

<p>LOX-1-V5-COS transfected cells were incubated with MβCD, GM6001, PMSF, Pepstatin A and the protease inhibitor cocktail (PI mix) (Calbiochem), as indicated (upper panel). Histogram shows the densitometric analysis expressed as percentage of reduction of sLOX-1 released in the presence of different protease inhibitors. 100% refers as sLOX-1 released in the presence of 5mM MβCD for 30 min (lower panel). Data in histograms represent the average ± SEM of three experiments, P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).</p

sLOX-1 release in COS cells.

<p>(A) LOX-1-V5-COS transfected cells were treated with filipin (5μM) and glimepiride (5μM) for 15, 30 and 60 min. Conditioned media were centrifuged at 100,000 g and the resulting pellets (P100, lanes 2–4) and supernatants (S100, lanes 5–13) were analyzed by Western blotting. Lane 1 (Extr.) shows total protein extract (5 μg) loaded as a positive internal control of electrophoretic mobility of full-length LOX-1 receptor. (B) Upper panel shows sLOX-1 amount released in cells treated with Filipin (5μM) and MβCD (5mM) for 30 min compared to sLOX-1 constitutively released from untreated cells at 1, 2 and 5 hours. Lower panel shows the densitometric analysis for sLOX-1 band intensity. Data in histograms represent the average ± SEM of three experiments, P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).</p

Soluble MMPs mediate LOX-1 shedding.

<p>(A) Fluorescence analysis of LOX-1-V5 transiently transfected COS cells treated without (Ctrl) or 5 mM MβCD or 30μM active MMP-2 or active MMP-1 for 1 h at 37°C. Images show Dil-labelled ox-LDL (red fluorescence) for 1 h at 4°C. Nuclei are blue stained with Hoechst 33342. Scale bar 20 nm. (B) Histogram shows the percentage of red positive cells in different treatments, counting Hoescht-stained nuclei (n≥150). Data represent the average ± SEM of two different experiments.</p

sLOX-1 release in EA.hy926 cells.

<p>(A) EA.hy926 cells were grown overnight in serum free medium in the absence (lanes 1–4) or in the presence (lanes 5–8) of ox-LDL (20μg/ml). MβCD was added to cells for the last 30 min incubation (lane 2, 3 and 8). GM6001 inhibitor was present for 30 min (lane 3) or overnight (lane 6). Western blot analysis of conditioned media was performed with anti-human LOX-1 receptor. (B) MTS assay performed on EA.hy926 cells plated at a density of 1,5x10⁴ cells/well and incubated overnight with different concentration of ox-LDL. Results are expressed as percentage of absorbance ± SEM compared to control cells.</p

LOX-1 release in exosomes.

<p>HEK-293#19 cells stably expressing LOX-1 were treated with filipin (5μM) and glimepiride (5μM) for 15, 30 and 60 min. Total protein extract (5μg) was loaded as a positive internal control of electrophoretic mobility (Extr.). Anti-V5 antibody was used to detect LOX-1-V5 protein and antibody directed against flotilin to verify equal protein loading.</p

Effect of receptor-ligand on LOX-1 shedding.

<p>Western blot analysis of media derived from LOX-1-V5 COS transfected cells incubated for 10 min with serum free DMEM (lane 1–3) or conditioned medium derived from EA cells (lane 4–6), in the presence of ox-LDL (A) and atorvastatin (B) at different concentrations, as indicated. Lower panels in (A) and (B) show a 6 times exposure of the gels.</p

EA.hy926 cells secretome induce LOX-1 shedding.

<p>(A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with DMEM (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).</p