Expression Levels Of The Predicted Proteins By Western Blotting

To evaluate the prediction accuracy of DAMAPEP, expression levels of the 10 proteins predicted with the 2.0 cutoff value were examined by Western blotting. Among the seven proteins predicted with increased expression in cancer cells, five proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2, were confirmed to have higher expression (Fig. 4a). For the other five proteins predicted with DAMAPEP, increased expression level of leukemia inhibitory receptor factor alpha was only detected in some cancer cell lines. For c-Kit and IL-2Rβ, no bands were observed in Western blots in any cell lines probably due to extremely low expression levels. The expression levels of Mos proto-oncogene and IκB-β were similar in both normal and cancer cell lines (data not shown). The expression of RAIDD was almost undetectable in the three normal cell lines. The expression of Rb p107, SRF, and Tyk2 in normal cells was between 15 and 40% of their expression in cancer cells. The expression of Rb p130 in normal cells was about 20% of its expression in cancer cells (Fig. 4b). Mean values of the normalized expression of RAIDD, Rb p107, Rb p130, SRF, and Tyk2 in three normal cell lines are 5 versus 61% (p = 0.0032), 26 versus 81% (p = 0.0041), 18 versus 69% (p = 0.0055), 25 versus 73% (p = 0.0033), and 30% (p = 0.0012), respectively (Fig. 4c). Those data confirm that RAIDD, Rb p107, Rb p130, SRF, and Tyk2 are indeed overexpressed in the tested cancer cells.

FIG. 4.

Validation of DAMAPEP-predicted resultsa, Western blotting confirms that five proteins have higher expression levels in cancer cells. Total cell extracts of the 10 breast cell lines were prepared as described under “Experimental Procedures.” Equal amounts of total lysates, as shown for actin, were loaded for Western blotting analysis. Proteins are labeled at the left of the gel, and tested cell lines are labeled at the topN1N2, and N3 represent three normal cell lines, ER+1ER+2, and ER+3 represent three estrogen receptor-positive carcinoma cell lines, and ER−1ER−2ER−3, and ER−4 represent four estrogen receptor-negative carcinoma cell lines. b, relative expression of five proteins in 10 breast cell lines. Western blot analysis for every protein was repeated at least three times. The average values for every protein in different cell lines are shown here with error bars corresponding to their S.D. c, box plot of the relative expression of five proteins in three normal cell lines and seven cancer cell lines using the data shown in b. The ranges of expression levels in the group of normal cell lines versus the group of cancer cell lines are shown side by side for every protein. The interquartile ranges are shown by boxes with the median values in filled trianglesN, normal;C, cancer; HQ, higher quartile; LQ, lower quartile.