Fig. 9

Elevated levels of 14-3-3ε and α-actinin 4 in CMT tissue-immersed PBS and sera of CMT-afflicted dogs. A Paired samples of CMT tissues (labeled C) and non-involved mammary gland tissues (labeled N) were obtained from nine female dogs afflicted with CMT. The tissue-immersed PBS samples were collected after one-hour incubation, and the tissue-released proteins were precipitated with TCA for immunoblotting analysis (10 μg of each sample) of 14-3-3ε and α-actinin 4. The CMT sample of patient #4 was replicated in two blots. Results were quantified and presented as fold changes of CMT over paired non-tumor samples. For patient#3, a fold change of CMT was calculated by comparing to the mean of all non-tumor samples. B Elevated levels of 14-3-3ε and α-actinin 4 were observed in CMT tissue-immersed PBS compared with non-tumor tissue-immersed samples. Data from all samples were normalized by the mean of total non-tumor samples. Statistical analysis was conducted using the Mann–Whitney U test. **p < 0.01. C Sera were collected from 17 female dogs afflicted with CMT and 15 age-matched healthy female dogs. Each serum sample (1 μL) was diluted in PBS, mixed with 4 × sampling buffer and subjected to SDS-PAGE with two gels. One gel was stained with Coomassie Brilliant Blue G-250, while the other was used for immunoblotting analysis. To quantify the levels of 14-3-3ε and α-actinin 4 in serum samples, the blot intensity of 14-3-3ε or α-actinin 4 was divided by the intensity of the entire lane of proteins stained with Coomassie Brilliant Blue for each sample. The resulting ratio was then normalized by the mean ratio of all healthy samples to acquire a normalized level for comparison. D Elevated levels of 14-3-3ε and α-actinin 4 were observed in sera from CMT-afflicted dogs compared with those in sera from healthy dogs. Statistical analysis was conducted with the Mann–Whitney U test. **p < 0.01