Fig 7.
Phosphorylation of Rpt6 at Ser120 is important in the PKA-mediated rescue effect on ST14A cells. ST14A cells were cotransfected with pcDNA3.1-Htt-(Q)109-hrGFP and pcDNA3-Rpt6-myc or pcDNA3.1-Rpt6S120A-myc for 24 h and then treated with the desired reagent(s) for another 48 h. Lysates collected for the indicated condition were used to perform the proteasome activity assay (A) and filter retardation assay (B and C). Data points represent the means ± SEM for 3 independent experiments. ST14A cells were cotransfected with pcDNA3.1-Htt-(Q)109-hrGFP and pcDNA3.1-Rpt6S120D-myc for 72 h. Lysates (30 μg per lane) collected for the indicated condition were subjected to a filter retardation assay (D). Insoluble Htt aggregates retained on the filter were detected using an anti-Htt antibody. The level of actin in the lysate was assessed using Western blot analysis and served as an internal control. A representative image of 3 independent experiments is shown. (E) Expression of hrGFP is shown in green. Nuclei were visualized using H33258 (blue). Representative pictures of 3 independent experiments are shown. Percentages of Htt aggregates containing cells were quantified using fluorescence microscopy. Data points represent the means ± SEM for 3 independent experiments. For each experiment, at least 130 transfected cells were scored.