These slides were imaged with a Sca nArray Express HT laser scanner. ScanArray Express software was used to quantify the spot fluorescence so never intensity from the scanned Imatinib 152459-95-5 images. Spot fluorescent data were then processed and analyzed using Protein Microarray Analysis Tool and ProMAT Cali brator, both Inhibitors,Modulators,Libraries of which are open source, freeware programs that we developed specifically for processing ELISA microarray data. Standard curves were fit to a four parameter logistic model and used to estimate the individual protein concentrations in each sample. RT PCR Analysis Inhibitors,Modulators,Libraries Quantitative assessment of mRNA expression was per formed by real time RT PCR. Total RNA was extracted using RNeasy kit.
The HMECs used in the quantitative RT PCR analyses Inhibitors,Modulators,Libraries were cultured and harvested in parallel with the cells used in the com parable protein experiments in the CM and cell lysates, although the RNA studies were conducted using Inhibitors,Modulators,Libraries cells raised in seperate dishes. Inhibitors,Modulators,Libraries Complementary DNA were synthesized from total RNA via reverse transcription using ImProm II reagents and oligo Inhibitors,Modulators,Libraries dT priming. The following primers were employed in the quantitative real time Inhibitors,Modulators,Libraries RT PCR Quantification of Erk/Akt activation Phosphorylated Erk1 and Akt levels of cell lysates were quantified by conventional ELISA techniques in a 96 well microplate. R D Systems DuoSet IC ELISA kits were used in these measurements, as described before. The manufactures protocols, including the lysis buffer, were followed in all the ELISA measurements.
Before each ELISA assay, protein concentrations Inhibitors,Modulators,Libraries of the cell lysates were measured using the Bicinchoninic Acid protein quantitation kit.
Data Analysis Inhibitors,Modulators,Libraries Concentrations of secreted proteins are presented in pg per ml of conditioned Inhibitors,Modulators,Libraries medium. The Inhibitors,Modulators,Libraries total volume of medium/dish was a constant within and across Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries stu dies. Concentrations of individual proteins in cell lysates are presented in ng per mg lysate protein. We used total secreted protein in the temporal stu dies for comparisons Inhibitors,Modulators,Libraries across the cells lines. An alterna tive approach would have been to use non stimulated cells as the control, and present the difference between stimulated and non stimulated cells.
This approach was not used because the cells were first fasted overnight in serum free medium.
To continue to deprive the cells of growth factors would have led to Paclitaxel price cell death during the remaining 24 hr study, and therefore was not a reason able option.
Rather, the approach used allowed us to compare Inhibitors,Modulators,Libraries cell lines EPZ-5676 solubility in parallel and directly demonstrate the involvement of HER2 or HER3 in protein secretion following HER1 activation. Statistical differences between cell lines following EGF stimulation and/or signaling pathway inhibition were initially determined by analysis of variance, and else then delineated using Fishers protected least significant dif ference test using StatView 5. 0. 1 software. A significance level of 0. 05 was used in all cases.