Figure 6

The ubiquitination and carboxyl-terminal region of Tat influence its activity towards microtubule assembly. (A) Anti-GFP immunoprecipitates and cell lysates were immunoblotted with anti-α-tubulin or anti-GFP antibodies. (B) 293 T cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone and treated with (+) or without (-) MG132. Anti-GFP immunoprecipitates and cell lysates were subjected to immunoblot analysis with antibodies against α-tubulin or GFP. (C) Schematic representation of the experimental workflow. Upon transfection of HeLa cells with the indicated plasmids, half the cells were boiled in SDS-PAGE sample buffer to obtain a mixture of soluble tubulin and polymeric fraction (microtubules) as total tubulin (T). The other half were subjected to the preparation of soluble tubulin (S) and polymeric fraction (P) as described in Materials and Methods. (D) HeLa cells were cotransfected with GFP-Tat101 and various ubiquitin mutants and treated with (+) or without (-) MG132. Protein samples were prepared as in (C) and immunoblotted with antibodies against α-tubulin or GFP. The ratios of polymeric fraction over soluble tubulin (P/S) were normalized against total tubulin levels and GFP intensity, and the relative folds were normalized to the untreated ubiquitin-K0 mutant transfection group and shown under the corresponding blots. (E) HeLa cells were transfected with GFP-Tat101, GFP-Tat86, GFP-Tat72 or GFP alone and treated with (+) or without (-) MG132. Protein samples were prepared as in (C) and analyzed as in (D). Cropped blots are used in this figure, and the gels were run under the same experimental conditions.