Figure 6. Nef is in protein complexes... | Rockefeller University Press

Figure 6.

$Figure 6. Nef is in protein complexes with autophagy regulator Beclin 1. (A) Macrophages were transfected with Nef-GFP and immunostained for Beclin 1. Arrows, perinuclear profiles exemplifying Nef and Beclin 1 colocalization. (inset) Peripheral colocalization. (B) 293T cells were transfected with Nef-GFP or GFP alone for 48 h. Lysates were immunoprecipitated either with Beclin 1 antibody and immunoblotted with Nef antibody (top) or with GFP and immunoblotted for Beclin 1 (bottom). Specific protein levels in cell lysates (input) and immunoprecipitates with IgG controls are shown. (C) Coimmunoprecipitation of Beclin 1 with Nef-Myc. 293T cells were transfected with Nef-Myc for 48 h, then cells were lysed and immunoprecipitation was performed using monoclonal Myc antibody or IgG control. Western blots were probed with Beclin 1 or Nef antibodies. The blots shown represent one of four independent experiments. (D) U937 cells were infected for 48 h with VSV-G–pseudotyped HIV. Cells were lysed and immunoprecipitated with Beclin 1–specific antibody or control IgG. Western blots of immunoprecipitated material were probed with Nef antibody. (E and F) 293T cells were cotransfected with Nef-GFP or GFP and Flag-hVPS34 for 48 h. Cells were fractionated into membranes (M) and cytosol (C) and immunoblotted with anti-Flag and Beclin 1 antibodies. (E) Immunoblots. (F) Quantification (ratios of membrane-associated hVPS34 to cytosolic hVPS34. Data indicate means; error bars indicate ±SEM. *, P < 0.05 (n = 3).$

Nef is in protein complexes with autophagy regulator Beclin 1. (A) Macrophages were transfected with Nef-GFP and immunostained for Beclin 1. Arrows, perinuclear profiles exemplifying Nef and Beclin 1 colocalization. (inset) Peripheral colocalization. (B) 293T cells were transfected with Nef-GFP or GFP alone for 48 h. Lysates were immunoprecipitated either with Beclin 1 antibody and immunoblotted with Nef antibody (top) or with GFP and immunoblotted for Beclin 1 (bottom). Specific protein levels in cell lysates (input) and immunoprecipitates with IgG controls are shown. (C) Coimmunoprecipitation of Beclin 1 with Nef-Myc. 293T cells were transfected with Nef-Myc for 48 h, then cells were lysed and immunoprecipitation was performed using monoclonal Myc antibody or IgG control. Western blots were probed with Beclin 1 or Nef antibodies. The blots shown represent one of four independent experiments. (D) U937 cells were infected for 48 h with VSV-G–pseudotyped HIV. Cells were lysed and immunoprecipitated with Beclin 1–specific antibody or control IgG. Western blots of immunoprecipitated material were probed with Nef antibody. (E and F) 293T cells were cotransfected with Nef-GFP or GFP and Flag-hVPS34 for 48 h. Cells were fractionated into membranes (M) and cytosol (C) and immunoblotted with anti-Flag and Beclin 1 antibodies. (E) Immunoblots. (F) Quantification (ratios of membrane-associated hVPS34 to cytosolic hVPS34. Data indicate means; error bars indicate ±SEM. *, P < 0.05 (n = 3).