L6pCDNA, L6 Flag ATM wt, GM 03189 and HeLa cell lines were cultured as described previously. Anti Mono and polyubiquitinylated conjugates mAb was bought from ENZO Life Sciences. Anti ATM mAb, anti Lamin W Ab, anti Matrin 3 Ab, anti PGAM1 mAb, anti PGK1 Ab, anti PKM2 Ab, anti Stat1 Ab and anti TPlastin mAb were obtained fromSanta Cruz Biotechnology. Anti hnRNP F H mAb was acquired by Abcam and anti compound library cancer GLRX1 Ab from R&D Systems Inc.. Anti BActin mAb and anti Tubulin mAb were purchased from Sigma Aldrich. ShATM construct and its get a handle on were described elsewhere. The proteasome inhibitor Z Leu?Leu?Leu al, DMSO, Trizma bottom, Urea, CHAPS, Iodoacetamide, DTT, Fibrinopeptide W, Ammoniumacetate, Methanol, Ethanol, Acetone and standard materials were purchased fromSigma. Sequence quality trypsin was purchased fromPromega. Water ultra gradient, Acetonitrile ultra gradient, TFAand Formic Acid were obtained by Romil. Chromoblastomycosis Protein extracts were obtained by lysing and sonicating cells in 6 M Urea, 100mM Tris pH 7. 5 and 0. Five minutes CHAPS. Protein concentration was based on the Bio Rad Protein Assay. Equal levels of proteins were resolved by 1 N SDS PAGE and blotted onto nitrocellulose membranes utilizing a Hoefer SemiPhor semidry exchange system. Blots were designed with the ECL plus chemiluminescences recognition system, extensively washed and, after incubation with horseradish peroxidase labeled goat anti mouse or anti rabbit or bovine anti goat Ab, incubated with the indicated primary antibodies. The band intensities were normalized and quantified with those of Tubulin using the image analysis software: ImageQuant TL. Three separate experiments natural compound library were done for each discovered protein. 2. 3. Expression analysis by nLC MSE Proteins extracted from 10?106 L6 and L6ATM cells, treated with 10 uM MG132 or 1:1000 DMSO for 2 hours, were quantified by Bio Rad analysis. Three different experiments were done and four protein pools were obtained, gathering 50 ug of protein fromeach experiment. Proteins pools were precipitated adding a of Methanol, Ethanol and Acetone, and redissolved in 6 MUrea, 100mMTris pH 7. 5. After reduction with 10mMDTT and alkylation with 20mM IAA, protein samples were digested 100:1 with collection grade trypsin at 37 C overnight. The reactionwas stopped with the addition of one last concentration of 0. 2 weeks TFA. Sampleswere dilutedwith 0. Fortnight FA, a few months ACN at a concentration of 0. 86 ug/ul, and 1. 72 ug of protein digestion were loaded on line for peptide separation. Previous of packing, 100 fmol/ul Saccharomyces cerevisiae Enolase digestion was included with samples as internal standard. Peptideswere caught on a um Symmetry C18 trapping column 180 um?20mm and separated using a 180 min RP slope at 300 nl/min on a UPLC System, utilizing a 1. 7 umBEH 130 C18NanoEase 75 um?25 cmnanoscale LC column.