Figure 2.

Protein expression in PyMT tumours. Randomly selected protein extracts from 9-week-old PyMT tumours were divided into groups according to tumour size (below or above 200 mm³) and diet with high (+) or normal (−) folic acid (FA). Control tissue: H, heart, K, kidney; B, wt FVB/N breast tissue. The sample marked with an asterisk was not included in the analysis. (A–C) Protein extracts from the individual tumours and control tissues were loaded on the gel, and immunoblot analysis was performed by using antibodies raised against the indicated proteins. Actin was used as a loading control. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis for the FR was performed under non-reducing conditions. (D) Immunoblot densitometry was performed, and the pSTAT3/STAT3, pSTAT3/actin and STAT3/actin ratios were determined. The values were normalised to protein extracts loaded on both gels serving as internal controls between gels. Statistical analyses using Student's t-test were performed (GraphPad Prism, La Jolla, CA, USA) with the following groups: FVB/N (N=3), PyMT <200 (N=6), PyMT<200+FA (N=5) and PyMT>200+FA (N=6). **P<0.01, *P<0.05 and NS, not significant.