FIG. 3.

Modification of N- or C-terminal residues affects hUbc9 protein detection in yeast. (A) ubc9-10 top1Δ cells (transformed with UBC9 vectors shown in Fig. 2) were galactose induced at 26°C or 36°C, and cell extracts were immunoblotted with antibodies recognizing h/yUbc9 (α Ubc9) or specific for yUbc9 (α yUbc9). Equal protein loading, assessed by tubulin staining (not shown), was mirrored by the nonspecific band (*) in the α Ubc9 panel. (B) hUbc9-Flag or hUbc9, immunoprecipitated from cell extracts with anti-Flag antibodies, was immunoblotted with α Ubc9. Cell extracts (input), supernatant (sup), and immunoprecipitated protein (IPα flag) samples are shown. (C) As in panel A, Ubc9 proteins expressed at 36°C were detected in immunoblots of ubc9-10 top1Δ cell extracts stained with α Ubc9. (D) As in Fig. 2, dilutions of ubc9-10 top1Δ cells transformed with the indicated YEpGAL1-UBC9 vector were spotted onto selective galactose plates with or without HU and incubated at 26°C or 36°C. (E) The formation of isopeptide linkages between Smt3-Smt3 and Ubc9-Smt3 was assayed in vitro at 36°C, resolved by polyacrylamide gel electrophoresis in the presence of DTT, and stained with SYPRO-Ruby. hUbc9 proteins were incubated with a 100-fold excess of yE1.