Figure 8

HDAC6 protects A3G from Vif-mediated ubiquitination and degradation in a BUZ domain-dependent manner. a Cell lysates from controls expressing A3G-3xHA, or lysates co-expressing A3G-3xHA with Vif, HA-wt-HDAC6 or HA-HDAC6-ΔBUZ were subjected to pull-down with Ni2⁺-NTA magnetic agarose beads to precipitate ubiquitinated proteins coupled to Ubiquitin-6xHis (overexpressed in all experimental conditions). Co-precipitated (co-P) extracts were immunoblotted with specific Abs directed against HA and HDAC6 to monitor co-precipitated ubiquitinated (-6xHis) A3G together with HDAC6 constructs. b Cell lysates from control scrambled or siRNA-HDAC6-treated cells, expressing A3G-3xHA with or without Vif, were subjected to pull-down with Ni2⁺-NTA magnetic beads to precipitate ubiquitinated proteins as above. Precipitated (P) extracts were immunoblotted with a specific Ab directed against HA to monitor ubiquitinated (6xHis) A3G. HDAC6/α-tubulin (to quantify silencing efficiency), A3G-3xHA/α-tubulin and (Ubiquitinated A3G-3xHA)/A3G-3xHA (to quantify the increase of A3G ubiquitination) ratios are shown. In a, b, input expression levels of overexpressed or silenced proteins, as well as endogenous HDAC6 and α-tubulin are shown. Data shown are representative of three independent experiments.