PTEN and Akt, phospho Akt. p42 44 MAP kinase and phospho p44 42 MAP kinase. p38 MAP kinase and phospho p38 MAP kinase. actin, EGFR, and phospho EGFR. Her2 and Her3. The membranes were then incubated with peroxidase conjugated secondary antibodies and protein was detected with enhanced chemiluminescence Western blotting detection reagents. These photos have been quan tified by measuring signal intensity utilizing National Insti tute of Well being Picture. The ratios of phospho EGFR EGFR, Her2 actin, Her3 actin, phos pho Akt Akt, PTEN actin, phospho p44 42 MAP kinase p44 42 MAP kinase, and phospho p38 MAP kinase p38 MAP kinase had been calculated from cells grown in both serum containing and serum free condi tions. Polymerase Chain Response Single Strand Conformation Polymorphism Analyses and Sequencing of Genomic DNA Genomic DNA samples were obtained from every cell line by proteinase K therapy and phenol chloroform extrac tion utilizing conventional protocols.
From each genomic DNA sample, exons 18, 19 and 21 of the EGFR gene and exon one and 2 on the K Ras gene had been amplified individually using the pertinent polymerase chain response prim ers applying the Gene Amp XL PCR kit. PCR conditions for genomic DNA analysis have been as follows. 40 cycles RAD001 price at 94 C for forty sec onds, 60 C for thirty seconds, and 68 C for 90 seconds, fol lowed by 68 C for 8 minutes. Fluorescein isothiocyanate labeled PCR solutions have been denatured, cooled on ice, and loaded on neutral 6% polyacrylamide gels with or with out 5% glycerol, as described previously. Just after electrophoresis, the gels have been analyzed with the FluorImager 595. Each DNA sample was examined with 3 primer pair combinations, with the M13 sequence additional in each and every case towards the appropri ate primer. PCR evaluation was performed as described above, with the resulting items staying purified and sequenced by fluorescence based automated sequencing.
FISH Examination Cells were fixed on slides in Carnoys fluid and incubated for thirty min at 60 C and 30 min at 37 C in two ? sodium chloride sodium citrate remedy 0. 1%Tween20 followed by ethanol dehydration. OC000459 The LSI EGFR CEP7 SpectrumGreen Probe was applied towards the slides which had been then incubated for 5 min at 75 C to the codenaturation of probe and chromosomal DNA. Hybrid ization proceeded overnight at 37 C, just after which the slides were washed in 50% formamide 2 ? SSC for 15 min, 2 ? SSC for ten min and two ? SSC 0. 1%Tween29 for five min at 47 C. The chromatin was counterstained with 46 diamidino 2 phenylindole. The fre quency of tumor cells with certain copy numbers of the EGFR gene and chromosome 7 had been estimated beneath a BX61 Olympus fluorescence microscope within a minimum of 200 nuclei. A gene amplifi cation was defined as gene chromosome per cell ratio 2 or 15 copies of EGFR per cell in 10% of analyzed cells. EGFR gene copy numbers were counted and were classi fied into six FISH strata by Hirshs criteria.