94Q S Q S 19Q S W1W Q 19Q TAP eluate 94Q S-1 W2 W1 W2 S-1A. Verification of TAP by Western blot (anti-FLAG). S: supernatant of cell lysate loaded to TAP Strep column; W1: sample collected from the flow-through of the Strep column during the washing step; W2: sample collected from the flow-through of the FLAG column during the washing step. A Supplementary Figures

S-1 SYPRO Ruby M 94Q M 19Q 55Q V mHtt wtHtt mHtt S-1B. SDS-PAGE separation of SF- Htt fusion proteins and their associated proteins. After elution of the Htt protein from the Strep and FLAG columns, the proteins that were associated with them was analyzed on a SDS-PAGE gel and stained with a SYPRO Ruby. M, protein molecular weight markers; size, in kD, is indicated on the left. Arrows indicate the bait proteins, which were verified by mass spectrometry. B

* * * S-2 S-2. Prevention of phosphorylation at Ser431 and S432 alters the toxicity of mHtt. HEK293 cells were transfected with indicated plasmid constructs and after 42 hours, the cells were treated with 200µM H 2 O 2 for 24 hour. The percentage (%) of cell viability was measured by the MTT method. * p < n = 4.

Actin 45kD Insoluble Htt (Stacking gel) Soluble Htt (Resolving gel) 95kD 94Q 94QS431 S/A S/D 94QS432 S/D S/A A S-3 S-3A. Prevention of phosphorylation at Ser431 and S432 alters mHtt aggregation. HEK293 cells were transfected with indicated plasmids and after 24 hours of transfection, the cells were collected and subjected Western blot using the antibodies as indicated on the right side of each panel.

* * * * B C S-3B. Quantification of the Western blot result showing protein levels of insoluble (mHtt aggregates found in stacking gel). * P < n = 3. S-3C. Quantification of the Western blot result showing protein levels of soluble mHtt (in resolving gel) (right). * P < n = 3. S-3