Molecular Analysis of a Recurrent Sarcoma Identifies a Mutation in FAF1

Figure 4

Protein overexpression. (a) 2D protein gel electrophoresis of lysates from muscle (upper left), tumor (upper right), and bone (bottom) in RIPA buffer. Red circles indicate the spots that were identified as overexpressed and were further analyzed by mass spectrometry. (b) Proteins identified in 2D gel electrophoresis as overexpressed in the tumor in comparison to muscle and bone were analyzed for their identity by mass spectrometry. The left column indicates the spot number corresponding to the 2D gel. The next column contains the protein name, followed by the accession number and a description of the protein function. The protein functions are grouped into structural, calcium homeostasis, and various others. (c) Western blot. 10 μg lysates of tumor, muscle, and bone in RIPA buffer were loaded per lane and electrophoresed on 10% SDS-polyacrylamide minigels with reducing, denaturing sample buffer. After transfer to PVDF membranes, they were probed for markers of cancer progression, including osteopontin, CD44, STAT3, and phospho-STAT3. Antitubulin served as a loading control. (d) RNA levels corresponding to the proteins found to be affected in the sarcoma. With four exceptions in the tumor-bone comparison (gray font), upregulated proteins are associated with increased RNA levels. STAT3 is not increased on the protein or RNA level. The reduced level of RB1 expression is consistent with the elevated DNA binding activity of E2F1.

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