Figure 3.

Normalized p62 levels, p62 flux, and p62 net flux analyzed by Western blotting in uninfected and HIV-infected macrophages. Primary human MDM were cultured as in the Materials and Methods section (Section 2) and left untreated (Untx) or treated with morphine and/or ART for 24 h with lysosomal inhibitors, NH4Cl and leupeptin (N+L) added in the last 4 h. (A) Representative Western blot with total protein loading control in uninfected cells. (B) p62 levels relative to total protein and normalized to the uninfected untreated control for each experiment. (C) p62 flux was quantified. (D) p62 net flux was quantified. (E) Representative Western blot with total protein loading control in HIV-infected cells. (F) p62 levels relative to total protein and normalized to the HIV-infected untreated control for each experiment. (G) p62 flux was quantified. (H) p62 net flux was quantified. (I) Normalized p62 values for infected MDM were corrected for the presence of HIV on average based on data in Supplementary Materials Figure S2 according to calculations described in the Materials and Methods section (Section 2), and comparisons were made between MOR and MOR+ART treatments in uninfected and infected cells. (J) p62 Flux values for infected MDM were corrected using the Normalized p62 values in (I) and compared to uninfected flux values. (K) p62 Net Flux values for infected MDM were corrected using the Normalized p62 values in (I) and compared to uninfected flux values. Error bars depict SEM, n = 5–10 independent experiments, * p < 0.05, ** p <0.01 one-sample t-test, *** p < 0.001 one-sample t-test.