Breast tumor kinase expression in human breast cancer cell lines
While studies in the MEF5-/- cells allowed us to characterize the role of Brk in STAT5b activation, we continued our studies in breast cancer cell lines as a means to investigate the biological relevance of Brk-mediated STAT5b activation. We determined the relative expression of Brk by analyzing mRNA and protein levels in a panel of frequently studied breast cancer cell lines. The total mRNA was isolated from each cell line, cDNA was generated, and primers specific for either Brk or β-actin were used for amplification [25]. In each sample, the relative expression of Brk mRNA was determined by normalizing to the amount of β-actin. The expression of Brk mRNA in the MDA-MB-468 cell line was set to 1 (Figure 4a) because this cell line expressed the lowest detectable amount of Brk mRNA. While BT-20 cells and T47D cells had the highest mRNA levels, SKBr3 cells and MDA-MB-468 cells expressed moderate levels, and BT-549 cells had no detectable Brk mRNA.
Figure 4  Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the cDNA was generated using iScript cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.   The amount of Brk protein in detergent cell lysates from each cell line was determined by immunoblotting. The relative expression of Brk protein was determined by normalizing to the nuclear import protein Ran, shown previously as a valid normalization control in breast cancer cell lines [28]. Again the amount of Brk protein in the MDA-MB-468 cell line was set to 1 (Figure 4b) because this cell line had the lowest detectable amount of Brk protein. Brk was highly expressed in BT-20 cells and T47D cells, was moderately expressed in SKBr3 cells and MDA-MB-468 cells, and was not detectable in BT-549 cells. Relatively high levels of Brk expression were seen in both the estrogen-receptor-positive T47D cells and the estrogen-receptor-negative BT-20 cells. Furthermore, while MDA-MB-468 cells, BT-20 cells, and BT-549 cells all express the EGFR, they expressed different amounts of Brk.