Figure 2.
HIV-1 Nef mimics the BafA1 to block autophagy in primary human astrocytes. PFHA were plated in 6-well plates as described earlier and transduced with Ad-Nef or Ad-Null when the cells were confluent. In this experiment, 3 times more (3 × 106 U) Ad-Nef was used to maximize Nef expression (Lane 3) and BafA1 (10 nM) treatment in untransduced cells was shown in Lane 4. Controls are shown in lane 1, untransduced PHFA and lane 2, Ad-null transduced PHFA Lane 2. Cells were harvested after 24 hours and analyzed by SDS-PAGE/Western Blotting as described before. Immunoblotting was performed using anti-HIV-1 Nef, anti-LC3B and anti p62 antibodies along with loading controls β-tubulin and GAPDH similar to shown in Fig. 1B. These results are representative of at least 3 different independent experiments.