Figure 1

The ubiquitination and carboxyl-terminal region of Tat regulate the stability of Tat. (A) Schematic representation and functional domains of HIV-1 Tat and schematic diagrams of GFP-tagged Tat101 and two truncated mutants. CRD, cysteine-rich domain; Core, conserved core region; Basic, region of basic amino acids; QRD, glutamine-rich domain; RGD, region of Arg-Gly-Asp sequence. (B and C) 293 T cells were cotransfected with GFP-Tat101 and His-Myc-tagged WT ubiquitin or the lysine mutants (K0, K29, K48, and K63) and treated with MG132. Anti-GFP immunoprecipitates and cell lysates were immunoblotted with anti-Myc or anti-GFP antibodies. (D) 293 T cells were cotransfected with GFP-Tat101 and His-Myc-tagged WT ubiquitin or the lysine mutants and treated with (+) or without (-) MG132. Cell lysates were subjected to immunoblot analysis with antibodies against GFP or α-tubulin. Anti-α-tubulin western blot was done as a loading control. (E) Quantification of the results in (D). Bars represent the relative ratios of GFP over α-tubulin levels normalized to the untreated ubiquitin-K0 mutant transfection group. (F) 293 T cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone and treated with (+) or without (-) MG132. Cell lysates were immunoblotted with anti-GFP or anti-α-tubulin antibodies. (G) Quantification of the results in (F). Bars represent the relative ratios of GFP over α-tubulin levels normalized to the untreated GFP vector transfection group. (H) Quantitative real-time PCR analysis of gene expression in 293 T cells transfected with GFP-Tat101, GFP-Tat86, or GFP-Tat72 and treated with (+) or without (-) MG132. GAPDH was used for normalization. Two-tailed Student’s t-test for all graphs. *P < 0.05, **P <0.01, ***P < 0.001; ns, not significant. Mean and standard deviations were derived from three independent biological replicates. Cropped blots are used in this figure, and the gels were run under the same experimental conditions.