Figure 4
From: HIV-1 Nef-induced cardiotoxicity through dysregulation of autophagy
Nef increased accumulation of protein aggregates in cardiomyocytes. (A) Representative confocal images show accumulation of ubiquitin in the cardiomyocytes expressing Nef. NRVCs were transduced with control Ad-null or Ad-Nef for 48 hours, fixed with 4% PFA, and stained with ubiquitin antibody. (B,C) Western blot analysis confirms that ubiquitin levels are increased in the Nef-treated cardiomyocytes (n = 4g/group, *P < 0.05, significant difference between Ad-null and Ad-Nef). (D,E) NRVCs were transduced with Ad-Nef or Ad-GFP for 48 hours and total protein lysates were analyzed by Western blot using SQSTM1/p62-specific antibody; p62 levels are significantly increased in Nef-expressing cardiomyocytes (n = 6/group, *P < 0.05, significant difference between Ad-GFP and Ad-Nef). (F) Representative confocal images indicate that Nef and p62 are colocalized in perinuclear aggregates in Nef-expressing cells. NRVCs were transduced with Ad-null or Ad-Nef for 48 hours and cells fixed with 4% PFA and stained with Nef- and p62-specific antibodies.
