Figure S3. Increased association of p62 and K63 ubiquitin with C6R mHTT. A COS-7 cells were cotransfected with mHTT aa 1-1212 (cleavable or C6R) or mHTT aa 1-586 and p62 as indicated. After immunoprecipitation of HTT, the ratio of co-immunoprecipitated p62 was quantified (normalized to input to control for transfection efficiency). B COS-7 cells were cotransfected with cleavable mHTT1-1212, C6R mHTT1-1212 and p62 as indicated and treated with MG132 to enforce autophagic degradation. Cycloheximide was added for the indicated periods of time and samples were analyzed by Western blot. Representative blots are shown as part of Fig. 3b. 2way-ANOVA HTT construct p=0.1451, time p<0.0001. C COS-7 cells were cotransfected with mHTT aa1-1212 (cleavable or C6R) and HA-tagged wt, K63 or K48 ubiquitin (allowing all, only K63 or only K48 linkage to target proteins) as indicated. After immunoprecipitation of HTT, the ratio of co-immunoprecipitated ubiquitin/HTT was quantified (normalized to input to control for transfection efficiency). Blots and quantification data with S.E.M. from a representative of 3 independent experiments are shown, number of technical replicates is shown as insets. Statistical significance was determined by 1way ANOVA with Tukey’s post-hoc correction (A), 2way-ANOVA with Bonferroni’s post-hoc correction (B) or Student’s t-test (D). *: p<0.05, **: p<0.01, ***: p<0.001. (TIFF 2089 kb
