94Q

S97

66

55Q

S

19Q

S W1 W2

97

66

55Q19Q

TAP eluate

94Q

S-1

W2W1 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-1SYPRO Ruby

M 94QM 19Q 55QV

29

45

66

116

205

97 mHtt

29

45

66

116

205

97

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 H2O2 for 24 hour. The percentage (%) of cell viability was measured by the MTT method. * p < 0.01. n = 4. 

Actin45kD

Insoluble Htt(Stacking gel)

Soluble Htt(Resolving gel)95kD

94Q94QS431

S/A S/D

94QS432

S/DS/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.

*

*

*

*BC

S-3B. Quantification of the Western blot result showing protein levels of insoluble (mHtt aggregates found in stacking gel). * P < 0.01. n = 3. S-3C. Quantification of the Western blot result showing protein levels of soluble mHtt (in resolving gel) (right). * P < 0.01. n = 3.

S-3