JCS040493 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Identification of the proteasomal machinery components in the protein complex(es) of ubiquitinated H-Ras. (A) HEK293 cells were transfected with pcDNA3.1-H-Ras-Myc and pEF-HA-Ub. Twenty-four hours after transfection, cells were treated with ALLN (50 µg/ml) for 12 hours before harvesting. Immunoprecipitation with anti-Myc resin was performed with 20 mg of total cell lysates. A small aliquot of immunoprecipitated lysates was used for confirmation of immunoprecipitation and polyubiquitination of H-Ras (left). Immunoprecipitated lysates were separated with SDS-PAGE and stained with Coomassie Blue (right). (B) The whole gel was separated into different areas by laser blade cut, and areas were analyzed with LC/MS-MS. The H-Ras-binding proteins related to the proteasome and ubiquitin were selectively presented.

• Supplemental Figure S2 -

  Fig. S2. Effects of β-TrCP overexpression in liver and colorectal cancer cells. (A) Effect of β-TrCP overexpression on overexpressed Ras-L61 and ERK activity in Chang liver cells. (B) Effect of β-TrCP overexpression on endogenous Pan-Ras and ERK activity in SW480 colorectal cancer cells. Both Chang and SW480 cells were transfected with a combination of pcDNA3.1, pMT3-H-Ras-L61, and pcDNA3.1-Flag-β-TrCP. Cells were harvested, and whole cell lysates were subjected to immunoblot analyses using anti-Flag, anti-β-TrCP, anti-Pan-Ras, anti-p-ERK, anti-ERK or anti-α-tubulin antibodies.

• Supplemental Figure S3 -

  Fig. S3. H-Ras specifically interacts with β-TrCP but not with FBW8, a β-TrCP analogue. HEK293 cells were transfected with pcDNA3.1-H-Ras-Myc, pcDNA3.1-Flag-β-TrCP or pcDNA3.1-Flag-FBW8. Cells were harvested and whole cell lysates were prepared. Lysates were subjected to immunoprecipitation with anti-Flag antibody, and the immunoprecipitated samples as well as the whole cell lysates were subjected to immunoblot analyses using anti-Flag, anti-H-Ras or anti-ERK antibodies.

• Supplemental Figure S4 -

  Fig. S4. Effects of Axin overexpression on the stability of Ras-L61. Chang or L929 fibroblast cells were transfected with combinations of vector, pMT3-Ras-L61 and pCS2-MT-Axin for 36 hours. Whole cell lysates were then analyzed by immunoblot analyses with anti-β-catenin, anti-Pan-Ras, anti-Axin or anti-α-tubulin antibodies.

• Supplemental Figure S5 -

  Fig. S5. Effects of Axin on the regulation of Ras ubiquitination. HEK293 cells were transfected with vector, Axin siRNA and/or HA-Ub expression plasmids for 36 hours. Where indicated, cells were treated with ALLN for 12 hours before harvest. The ubiquitination assay and immunoblotting were performed as in Fig. 5.

• Supplemental Figure S6 -

  Fig. S6. Effect of β-TrCP on regulation of K-Ras, N-Ras or H-Ras. HEK293 cells were transfected with pcDNA3.1, pcDNA3.1-H-Ras, pcDNA3.1-K-Ras or pcDNA3.1-N-Ras-Myc together with or without pcDNA3.1-Flag-β-TrCP for 36 hours. The whole-cell lysates were analyzed by immunoblot analyses with anti-β-TrCP, anti-Myc or anti-α-tubulin antibody.