ATPase activity of Pof1p and its physical interaction with Ubc7 (ubiquitin conjugase 7) protein. (A) Hydrolysis of ATP as measured by the PiPer™ Phosphate Assay Kit (Invitrogen). (-) ATP represents the result of the reaction in the absence of ATP; (-) POF1 represents the result of the spontaneous ATP hydrolysis in the absence of Pof1p; the lane labeled "Pof1p + ATP" contains the complete reaction. The assays were performed at 37°C for 1 h. (B) Western blot analyses of Ubc7p using the commercial antibody Ube2G2 (Abcam). The fractions were obtained from co-immunoprecipitation assays using Pof1p polyclonal antibody from the following protein extract: WT = wild type; Δpct1 and Δpof1. The asterisk shows the Pof1p-Ubc7p complex. CE = total soluble wild type cell extract; IgG LC = IgG light chain. The arrow points Ubc7p dimer.