Fig. 7.

Tat Ser-46 mutations on its ubiquitination. a Tat S46A mutation decreased Tat ubiquitination. 293T cells were co-transfected with pCI-His-hUbi plasmid and WT Flag-Tat, Flag-Tat S16A, Flag-Tat S46A expressing plasmids. The cells were lysed at 48 h posttransfection. His–Ub conjugated proteins were extracted in guanidine denaturing buffer and purified on Ni–NTA agarose beads as described in Methods. Proteins were eluted from the beads and resolved on 12% Tris-Tricine SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes and immunoblotted with anti-Flag antibodies which detected monoubiquitinated Tat. Loading controls were obtained by resolving a portion of the total lysate on 12% SDS-PAGE. Lower panel show quantification as a mean of three independent measurements ± SD. Unpaired t test was used to test statistical significance. *p ≤ 0.01. b Tat S46D mutation decreased Tat ubiquitination. 293T cells were transfected and processed as in a except WT Flag-Tat, Flag-Tat S16D, Flag-Tat S46D and Flag-Tat K71A expressing plasmids were used. Lower panel show quantification