ed was raised against the N terminal domain, recognizing also mutated and expressed p53 proteins. p53 mutation analysis Genomic DNA was isolated using the QIAamp DNA Micro Kit according to the manufacturers instruction. Amplification of p53 exons 2 11 was performed using primers and protocols slightly modified from previous studies. PCR was carried out in a 25 ul reaction mixture containing 1�� PCR Buf fer, 1. 5 2. 5 mM MgCl2, 12 ng ul gDNA, 0. 4 mM dNTP Mix, 0. 4 uM forward and reverse primers and 1. 25 U Taq DNA polymerase. The PCR was performed with the following conditions, 94 C for 4 min, 40 cycles con sisting of 94 C for 30 sec, 53 65 C for 30 sec and 72 C for 30 sec, followed by 72 C for 7 min. PCR products were purified using the QIAquick PCR Purification Kit according to the manufac turers protocol.

Sequencing was performed using Big Dye Terminator v1. 1 Cycle Sequencing Kit according to the man ufacturers instruction. The reactions were performed in 20 ul reaction mixture consisting of 3 5 ng PCR pro duct, 0. 16 uM forward or reverse primers, 20% BigDye Carfilzomib Ready Reaction Mix and 1�� Big Dye Sequen cing Buffer. A positive control with a 20 ul reaction mixture containing 5% pGEM 3Zf double stranded DNA control Template, 5% 21 M13 Control Primer, 20% BigDye Ready Reaction Mix and 1�� Big Dye Sequencing Buffer was included. The PCR was performed with the following conditions, 96 C for 1 min, 24 cycles consisting of 96 C for 10 sec, 50 C for 5 sec and 60 C for 4 min. DNA was precipitated with ethanol containing 5 mM EDTA and 120 mM sodium acetate, dissolved in formamide and denatured for 5 min at 95 C.

Capillary electrophoresis was performed using the ABI PRISM 310 Genetic Analyzer. The Sequencing Analysis Software V 5. 2 was used to analyze the collected electropherogram traces and sequencing infor mation. The p53 sequence of the GenBank database with accession number NC 000017. 9|NC 000017, c7531642 7512445 was used as reference. RNA isolation and cDNA synthesis Total RNA isolation was performed using the RNeasy Mini Kit according to the manufacturers instruction. For cDNA synthesis, a 9 ul reaction mixture containing 200 ng total RNA, 1 ul yeast RNA and 2 ul Hexanucleotide Mix was incubated for 2 min at 70 C and 10 min at RT. A sec ond 11 ul reaction mixture containing 4 ul First Strand Buffer, 2 ul DTT, 1 ul dNTP Mix and 1 ul M MLV RT, was added and incubated for 1 h at 37 C.

The M MLV RT was inactivated for 5 min at 95 C. For reverse transcription of Universal Human Reference RNA, the calibrator of qRT PCR, 300 ng RNA was employed in an appropriate volume. HS 1 associated protein X 1, Hax 1, is a 35 kDa pro tein with two Bcl 2 homology domains that was identified in a yeast two hybrid screen where it was found to interact with HS 1, a Src kinase substrate. Hax 1 is ubiquitously expressed in most tissues and is reported to be localized in mitochondria as well as the endoplasmic reticulum and nuclear membrane. Mutations identified in the human