Home > Internet > MS-275 - About How Along with Why Users Can Easily Reap Benefits Out Of That

MS-275 - About How Along with Why Users Can Easily Reap Benefits Out Of That

Added: (Tue Nov 14 2017)

Pressbox (Press Release) - We stably over-expressed in RAW macrophages the FERM (1�C310) domain of ezrin fused to GFP (FERM-GFP). This domain binds to the endogenous ezrin protein, thereby blocking its activity. As a control, we used a RAW macrophage cell line (Ezr-GFP) stably expressing the full-length molecule of ezrin fused to GFP at the C-terminal of the molecule (Ez-GFPc). We incubated these cells with 3 ?m avidin-coated latex beads and fixed the cells at 2 h post-internalization. We calculated the percentage of maturing phagosomes (positive GDC-0449 price for LAMP-2) that displayed an F-actin labeling (Figure 2A,B). We found a significant twofold reduction in F-actin-positive phagosomes (Figure 2C; 16 �� 3.5%, p <0.01) in FERM-GFP cells in comparison to non-transfected or Ezr-GFP-expressing macrophages (31 �� 4.5%). Interestingly, we observed in Ezr-GFP cells that in most of the cases the actin foci detected on the phagosomal membrane were localized at the contact site between docked lysosomes and the phagosome (Figure 2A, inset), though the ezrin�CGFP signal was not as easily visible as labeling of ezrin by specific antibodies (Figure 1A). The FERM-GFP domain was also observed localized on some late phagosomes negative for F-actin (Figure 2B, inset). We have previously shown that the binding of the purified MS-275 clinical trial FERM domain of ezrin on isolated 2 h phagosomes inhibits their F-actin assembly activity in vitro(5). The reduction in the percentage of actin-positive phagosomes in FERM-GFP-expressing cells suggests that this domain displays a DN effect, not only in vitro but also in cells. We therefore investigated by WB whether this domain was recruited on late phagosomes. Quinapyramine Isolated 2 h phagosomes from Ezr-GFP and FERM-GFP cells were lysed and equal amount of phagosomal proteins were fractionated by SDS�CPAGE. We detected both the Ez-GFPc protein and the FERM-GFP domain on phagosomes from Ezr-GFP and FERM-GFP cells, respectively (Figure 2D), showing that the ezrin over-expressed constructs were recruited on late phagosomes in cells. Endogenous ezrin was found in similar amount in both phagosome types, suggesting that the FERM-GFP construct did not display a DN effect by displacing the endogenous ezrin but rather by blocking its activity. Accordingly, we found that the level of N-WASP on FERM-GFP-positive phagosomes was reduced compared to Ezr-GFP-positive phagosomes (Figure 2D), suggesting that the presence of the FERM domain partially inhibited the recruitment of N-WASP on late phagosomes. This likely caused the reduction in the phagosome F-actin assembly that we observed in FERM-GFP cells compared to Ezr-GFP cells (Figure 2B). Importantly, we found that Rab7, a GTPase crucial for phagosome maturation and in particular for fusion with late endosomal compartments, was found in equal amount in FERM-GFP and Ezr-GFP phagosomes, showing that its recruitment was not affected by the FERM-GFP domain (Figure 2D).

Submitted by:
Disclaimer: Pressbox disclaims any inaccuracies in the content contained in these releases. If you would like a release removed please send an email to remove@pressbox.co.uk together with the url of the release.