For experiments involving over expression of AnxA6, former the coding sequence of AnxA6 variant 1 Inhibitors,Modulators,Libraries was amplified from plas mid pCMV Sport6 AnxA6. The fragment was cloned into Hind III and Xho I lin earized pCMV 3Tag 8, and the construct used to transfect the AnxA6 low HCC1806 BCCs using Fugene6 transfection reagent. The transfected cells herein designated 1806 Anx6 and 1806 EV were selected with hygromycin, cloned as above and expanded in continued hygromycin selection. Immunofluorescence microscopy Cells were plated sparsely on glass cover slips and allowed to grow until they formed colonies of a few cells. Cells were then serum starved overnight and treated with or without EGF for 5 min. Inhibitors,Modulators,Libraries Indirect immunofluorescence staining was performed as previously described using pEGFR antibodies and FITC conjugated secondary anti bodies.

Cover slips Inhibitors,Modulators,Libraries were mounted with ProLong Gold anti fade containing DAPI. Images were captured using a Nikon A1R confocal microscope with 60 oil immersion objectives and analyzed using the NIS software. Cell surface biotinylation and Western blotting Cells were grown in complete medium until they were 70% confluent, then serum starved for 24 h and treated with or without EGF for the indicated time points. Following treatment, cells were washed twice with ice cold phosphate buffered saline pH 7. 4, with 0. 5 mM Ca2 and 1 mM Mg2 and then incu bated with Sulfo NHS biotin for 30 min at 4 C. Unreacted biotin was quenched with ice cold 100 mM glycine in PBS for 15 min at 4 C. Whole cell extracts were prepared in TNE lysis buffer.

Inhibitors,Modulators,Libraries Biotinylated proteins isolated using Streptavidin agarose beads and whole cell extracts were used for the detection of cell surface and total cellular EGFR respectively by West ern blotting as previously described. Immuno reactive bands were visualized by enhanced Inhibitors,Modulators,Libraries chemilu minescence and quantified using NIH Image J software. Activation of EGFR and downstream signaling assays Breast epithelial and BCCs were cultured until they were 70% confluent then serum starved overnight and treated with 50 ng ml EGF in Hanks bal anced salt solution for the indicated time pe riods. The EGF treated cells were scraped in ice cold PBS and total cell lysates prepared as described previ ously. EGFR activation was detected by immunoblot ting with anti EGFR and antibodies to total EGFR.

Activation of downstream signaling cascades was determined by Western blotting using ant pErk1 2 and anti pAkt. Immunoblotting Trichostatin A 58880-19-6 with antibodies to either anti Erk2, anti GAPDH or anti B tubulin were used as the loading controls. Immuno reactive bands were revealed by ECL, scanned and quan tified using NIH Image J software. Activation levels were determined as the ratios of phosphoprotein to the total protein or loading controls. Cell proliferation assays The effects of AnxA6 depletion and TKIs on cell growth were performed in 24 well plates in triplicates using 1 x 104 cells well, as previously described.