JCS050013 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig S1. Pak2 immunoprecipitates Htt. Mouse brain lysate was immunoprecipitated with anti-Myc antibody as a control (NS, lane 2), or anti-Pak2 antibody (C-19, Santa Cruz) (lane 3) and blot was probed with anti-Htt antibody (top) and anti-Pak2 antibody (bottom) respectively. 6% of total lysate was loaded in lane 1. FL-htt: full-length Htt.

• Supplemental Figure S2 -

  Fig S2. Htt binds to Pak2 in kinase-independent manner. Pak2-vector (lane 1), Pak2-Flag-Htt588 (lane 2), Pak2-402E−Flag-Htt588 (lane 3) and Pak1-278R−Flag-Htt588 (lane 4), were transfected into HeLa cells. Cell lysates were immunoprecipited with anti-Flag antibody. Blots of immunoprecipitates were probed with anti-Pak2 antibody (panel 1), or anti-Flag (panel 3). Total lysates were probed with anti-Pak2 antibody (panel 2) and anti-Flag antibody (panel 4).

• Supplemental Figure S3 -

  Fig S3. Increasing levels of Htt decrease Pak2 cleavage. Data from three independent experiments. y-axis: ratio of cleaved Pak2 to full-length Pak2 (CL/FL Pak2); x-axis: the amount of Htt588. The ratio of cleaved Pak2 to full-length Pak2 was set as 1 in the case of no Htt588. Please note that the total amount of transfected DNA is kept constant in all cases.

• Supplemental Figure S4 -

  Fig S4. Proteasome inhibitors epoxomycin and lactsystin enhance cell death.

  (a) HeLa-Tet on cells were treated with DMSO, epoxomycin (1uM), lactacystein (10uM) in triplicate. After 24 hours, cell death was assessed with Trypan blue staining. Comparisons were performed with Student’s t-test, and errors bar are SD. *P₁=0.0222; *P₂=0.0051.

  (b) pEGFP was co-transfected into HeLa cells with Pak2-empty vector, or Pak2-Htt552, respectively (0.4:1.0:1.8). After 24 hours, cells were treated with epoxomycin or lactasystin for 14 hours. The transfected cells were then fixed and cell death of GFP-positive cells was evaluated by scoring apoptotic nuclear morphology. Samples are in triplicate. Comparisons were made with Student’s t-test. *: P₁=0.038; *: P₂=0.0077.

• Supplemental Figure S5 -

  Fig S5. Similar expression levels of different Htt variants. pCI vector, Htt120, Htt190, Htt315, Htt552, Htt588 were co-transfected into HeLa Tet-on cells with EGFP as a transfection/loading control. After 24 hours, cells were treated with TNFα+CHX for 4 hours, then the transfected cells were harvested and subjected to SDS-PAGE and western blot. The blot was detected with anti-Flag antibody for Htt variants (top panel). Note that Htt588 and Htt552 exhibit three bands and two bands, respectively, due to caspase cleavage. Further aliquots were subsequently subjected to SDS-PAGE and detected with anti-EGFP antibody (bottom panel). EGFP is transfection efficiency control. Note that Htt190 and Htt315 are not expressed at lower levels than Htt 552 and Htt588.

• Supplemental Figure S6 -

  Fig S6. Wild-type Htt binds to mouse Pak2 (mPak2) and prevents its cleavage in cells.

  (a) Myc-mPak2 was transfected along with an empty vector or Htt588 into HeLa cells. After 20 hours, cells were harvested and cell lysates were immunoprecipited with anti-Flag antibody. Blots of immunoprecipitates were probed with anti-Pak2 antibody (panel 1), or anti-Flag (panel 2). Total lysates were probed with anti-Pak2 antibody (panel 3) and anti-Flag antibody (panel 4).

  (b) Myc-mPak2 was transfected along with empty vector (lanes 1, 3) or Htt588 (lanes 2, 4) into HeLa cells. After 20 hours, transfected cells were treated with CHX (lanes 1-2) or TNFα and CHX (lanes 3-4), respectively, for 6 hours. The cell lysates were subjected to SDS-PAGE and blotting with anti-Myc antibody (top) to detect full-length Pak2 and N-terminal truncated Pak2 tPak2 (N-) and anti-Flag antibody (bottom). Blot is representative of data from three independent experiments.

• Supplemental Figure S7 -

  Fig S7. Model of Htt prevention of Pak2 cleavage. In the presence of death stimuli, activated caspases cleave Pak2 at D212 and release C-terminal kinase active form, tPak, which is toxic to cells. The binding of Htt to Pak2 prevents caspase-mediated cleavage and cell death.