Cells: human U251 glioblastoma cell line

Experiment:  

The U251 cell line was obtained from American Type Culture Collection (ATCC). Cells were cultured in improved minimum essential medium (IMEM) (Invitrogen, Carlsbad, CA) 
supplemented with 10% fetal bovine serum (Atlanta Biologicals, Inc., Lawrenceville, GA). Cells were grown until 90% confluence and then harvested to prepare RNA and protein extracts for further 
analyses.

Cells were transfected with a GFP lentivirus construct together with random shRNA. 

To prepare protein extracts, cells were re-suspended in an equal volume of lysis buffer (50mM Tris pH 8.0, 50mM NaCl, 1mM EDTA and 1 tablet of Complete Proteinase Inhibitor (Roche)/10ml) 
and incubated on ice for 30 minutes. Cells were then lysed with a dounce homogenizer and the soluble protein extracts obtained after centrifugation.

The pellet re-homogenized with dounce homogenizer and via passage through syringe (Pellet fraction). 


CYS modification: 

YES

Mass spectrometry:

LC-MS/MS on LTQ-Orbitrap

Analysis:

SEQUEST (Bioworks)
Peptide/ProteinProphet v4.0