Cytokeratin 14

Total protein was extracted with RIPA buffer and subjected to SDS-PAGE analysis prior to transfer to a PVDF membrane. Blots were probed with specific antibodies to DEK (BD Bioscience, San Jose, CA); Ron (C-20), Wnt10b [H70], and cyclin D1 [72-13G] (Santa Cruz, Dallas, TX); alpha-tubulin (Sigma), Actin C4 (gift of James Lessard, Cincinnati Children’s Hospital); Axin 2, MMP-2, and MMP-9 (Abcam, Cambridge, MA), cytokeratin 14 (Covance, Princeton, NJ), and GSK3β, phospho-ERK1/2, total ERK1/2, and TCF1 (Cell Signaling, Danvers, MA). Protein from primary mammary epithelial cells was collected from 18 week old nulliparous, non-tumor bearing Dek^+/+ females by isolating the cells through collagenase digestion and differential centrifugation as described above immediately prior to the addition of lysis buffer. For secreted proteins, 3×10⁶ cells were plated on 10cm dishes in complete media for 2 hours cells then washed in PBS and overlayed with media containing 1% FBS for 24 hours. Six milliliters of conditioned media was collected from each cell line and concentrated to 100 μl using Amicon Ultra-4 centrifugal filters with Ultracel-3 membrane (Millipore, Billerica, MA) then 2 μl was diluted into 18 μl of serum free media prior to western blotting. Western blots were analyzed by densitometry using Image J.^{70 (link)}