Levels of the Carfilzomib HER receptors in these cell lines were quantified selleck screening library as described before. Results from this analysis showed that there are approximately 2×105 HER1, 3×104 HER2 and 2×103 HER3 receptors on each parental HER1 cell, 1. 5×105 HER1, 6×105 HER2 and 2×103 HER3 on each HER2 till cell, and 2×105 HER1, 3 104 HER2 and 2. 8 104 HER3 on each HER3 cell. It has been reported that the well characterized breast cancer cell line SK BR3 express about 2×105 HER1, 6×105 HER2 and 1×104 HER3 receptors. Inhibitors,Modulators,Libraries Based on these reports and another evaluation of HER receptor concen trations in breast cancer cells, our HER2 or HER3 cell lines express HER2 or HER3, respectively, Inhibitors,Modulators,Libraries at levels that Inhibitors,Modulators,Libraries are comparable to those found in breast cancer cells that overexpress these receptors.

Cell Culture and Treatment Only cells that were less Inhibitors,Modulators,Libraries than fifteen passages from the original frozen stock were used in this study. Each cell line was seeded Inhibitors,Modulators,Libraries at 3. 0×105 cells per well of 6 well plates in DFCI 1 culture medium, and were allowed to grow to Inhibitors,Modulators,Libraries confluence prior to treatment. Before EGF treatment, cultured cells were fasted for 14 18 h in serum free, DFCI 1 medium that lacked all supplements except 0. 1% bovine serum albu min. Cells were then washed twice with buffered saline, pH 7. 2 7. 4, followed by the addition of 1 ml fresh serum free medium with Inhibitors,Modulators,Libraries 12 ng/ml EGF added. Acti vated cells were incubated at 37 C for up to 24 h and samples were collected at fixed time points after EGF addition.

Immediately Inhibitors,Modulators,Libraries prior to harvesting, cells were Inhibitors,Modulators,Libraries chilled by placing the culture dishes on ice.

Sample Collection and Processing Conditioned medium from the cultured cells was transferred to Inhibitors,Modulators,Libraries microcentrifuge Inhibitors,Modulators,Libraries tubes, and centrifuged at 2000 g for 5 min at 4 C in order to remove Inhibitors,Modulators,Libraries any particu lates or cell debris. An aliquot of each supernatant was then transferred to another tube that contained a 1/10th volume of 1% casein and green fluorescent protein in PBS, such that the final con centration of the green fluorescent protein was 100 pg/ ml. Green fluorescent protein was used as the antigen in an internal calibrant assay based on a sandwich ELISA.

The fluorescent signal from the capture antibody Inhibitors,Modulators,Libraries in this assay selleck kinase inhibitor was used for data normalization using a custom bioinformatics program, ProMAT Calibrator, as described Inhibitors,Modulators,Libraries below.

The cells on these culture plates were then washed twice with cold PBS, and harvested by add ing 200��l of lysis buffer.

Cell lysates were collected and centrifuged at 18,500 g for 10 min. The protein concentration Inhibitors,Modulators,Libraries Sunitinib purchase of cell lysates was measured using the Bicinchoninic Acid protein quantitation kit and averaged 1. 6 0. selleck bio 3 mg/ml. Since 200��l of lysis buffer was added, we estimate that about 0. 32 mg of protein was collected from each plate. In certain experiments, cultured cells were pre incu bated with a single inhibitor for 1 h prior to EGF addition.