Fig. 1

Effect of F07#13 on Tat degradation. a Following transfection into Jurkat cells, samples were collected and lysates were prepared for immunoprecipitation. Anti-Flag Ab was used for IP overnight, Protein A/G added the next day, washed, and samples were run on a gel and analyzed by Western blot for presence of Tat (α-Tat polyclonal Ab). Lanes 1 and 2 serve as control input transfected lysates (1/10) prior to IP. b Jurkat cells were transfected with 89.6 plasmid (20 μg) and CMV-Flag-Tat₁₀₁ (20 μg) and 24 h later samples were treated with 0.01, 0.1, and 1 μM of F07#13 for an additional 48 h (a total 72 h). Cells were pelleted and washed, and lysates were run on a 4–20% Tris–glycine gel followed by Western blot with α-Flag antibody, followed by α-actin as a control. An IP with α-Flag antibody was run on a gel and probed with α-ubiquitin antibody. Densitometry was performed for each lane. c Cells were transfected with both 89.6 and Tat vector, followed by treatment with F07#13 (48 h; 1 μM) and two other inhibitors, MG132 (10 ng/mL) and a de-ubiquitin USP7 inhibitor (P5091; 3 μM), for 24 h and then separated on 4-20% Tris–glycine gel followed by Western blot with α-Flag antibody, α-ubiquitin antibody, and α-actin. Densitometry was performed to visualize changes in protein expression. The quantitation of 5 distinct bands in each lane was performed and summed to obtain total densitometry counts