American Society of Hematology

Figure 5

$Figure 5. Nef L164-165L motif is required for interaction with AP1 and inhibition of macrophages phagocytosis. (A) GST, GST-Nef WT, or GST-NefL164-165A bound to glutathione sepharose beads were incubated with RAW264.7 macrophages lysates. Bound material (top panel) and total lysates (bottom panel) were subjected to Western blotting with anti-AP1 antibodies. Molecular weight markers are in kilodaltons. (B) RAW264.7 macrophages transiently expressing either Nef STOP-HA or Nef WT-HA or NefL164-165A-HA were lysed and immunoprecipitated with anti-HA antibodies. The precipitates and total lysates were revealed by Western blotting with anti-AP1 antibodies and after stripping with anti-HA antibodies. Molecular weight markers are in kilodaltons. (C) RAW264.7 macrophages transiently expressing either Nef WT-GFP or Nef G2-3A-GFP were fractionated to obtain a cytosolic and a membrane fraction. Then each fraction was immunoprecipitated with anti-GFP antibodies. The precipitates and total lysates were subjected to Western blotting with anti-AP1 antibodies and after stripping with anti-GFP antibodies. Total lysates were also revealed with anti–transferrin receptor antibodies (as marker for the membrane fraction) or with anti-Rho GDI alpha antibodies (as marker for the cytosolic fraction). The cytosolic fraction was contaminated with proteins of membrane origin. Molecular weight markers are in kilodaltons.$

Nef L164-165L motif is required for interaction with AP1 and inhibition of macrophages phagocytosis. (A) GST, GST-Nef WT, or GST-NefL164-165A bound to glutathione sepharose beads were incubated with RAW264.7 macrophages lysates. Bound material (top panel) and total lysates (bottom panel) were subjected to Western blotting with anti-AP1 antibodies. Molecular weight markers are in kilodaltons. (B) RAW264.7 macrophages transiently expressing either Nef STOP-HA or Nef WT-HA or NefL164-165A-HA were lysed and immunoprecipitated with anti-HA antibodies. The precipitates and total lysates were revealed by Western blotting with anti-AP1 antibodies and after stripping with anti-HA antibodies. Molecular weight markers are in kilodaltons. (C) RAW264.7 macrophages transiently expressing either Nef WT-GFP or Nef G2-3A-GFP were fractionated to obtain a cytosolic and a membrane fraction. Then each fraction was immunoprecipitated with anti-GFP antibodies. The precipitates and total lysates were subjected to Western blotting with anti-AP1 antibodies and after stripping with anti-GFP antibodies. Total lysates were also revealed with anti–transferrin receptor antibodies (as marker for the membrane fraction) or with anti-Rho GDI alpha antibodies (as marker for the cytosolic fraction). The cytosolic fraction was contaminated with proteins of membrane origin. Molecular weight markers are in kilodaltons.

This Feature Is Available To Subscribers Only