A modified real competitive PCR (mrcPCR) was performed to detect the FAK-copy-gain of breast cancer cell lines as previously described [10 (link)].
Reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (real-time qPCR) were performed according to methods described previously [39 (link)]. GAPDH was used as a control for the level of expression. The primers utilized were as follows: FAK (forward: 5’-TGA GAT CCT GTC TCC AGT CTA CAG-3’, reverse: 5’-CAG TAC CCA TCT ATT AGG TCA GCC-3’), GAPDH (forward: 5’-TGA TGA CAT CAA GAA GGT GGT GAA-3’, reverse: 5’-TCC TTG GAG GCC ATG TGG GCC AT-3’).
Western blotting was performed according to a method described previously [39 (link)]. Anti-FAK (#05-537), anti-pFAK (Y397, #05-1140), and anti-cleaved caspase-3 (#AB3623) were purchased from Millipore (Billerica, MA, USA), and anti-β-actin antibody (#A2228) was acquired from Sigma-Aldrich. Anti-Caspase-3 (#9662), anti-AKT (#9272), and anti-pAKT (S473) antibodies were obtained from Cell Signaling (Danvers, MA, USA).
Reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (real-time qPCR) were performed according to methods described previously [39 (link)]. GAPDH was used as a control for the level of expression. The primers utilized were as follows: FAK (forward: 5’-TGA GAT CCT GTC TCC AGT CTA CAG-3’, reverse: 5’-CAG TAC CCA TCT ATT AGG TCA GCC-3’), GAPDH (forward: 5’-TGA TGA CAT CAA GAA GGT GGT GAA-3’, reverse: 5’-TCC TTG GAG GCC ATG TGG GCC AT-3’).
Western blotting was performed according to a method described previously [39 (link)]. Anti-FAK (#05-537), anti-pFAK (Y397, #05-1140), and anti-cleaved caspase-3 (#AB3623) were purchased from Millipore (Billerica, MA, USA), and anti-β-actin antibody (#A2228) was acquired from Sigma-Aldrich. Anti-Caspase-3 (#9662), anti-AKT (#9272), and anti-pAKT (S473) antibodies were obtained from Cell Signaling (Danvers, MA, USA).
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