(2007) A curated compendium of phosphorylation motifs. and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants guarded cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. for 15 min and the total protein content was quantified by a BCA assay. To reduce nonspecific binding the supernatants were preincubated with protein G-Sepharose beads at 4 C for 1 h. Following a brief centrifugation, the supernatants were incubated with anti-HA rat-specific antibody with gentle agitation overnight at 4 C, followed by incubation with protein G-Sepharose beads at 4 C for 2 h. After a brief centrifugation the beads were washed three times in Azilsartan (TAK-536) lysis buffer, and resuspended in Laemmli sample buffer made up of no -mercaptoethanol and boiled for 5 min to release bound proteins. Proteins were loaded onto SDS-PAGE (10, 12, or 4C20% gels) and subjected to immunoblotting. For immunoblot and mass spectrometry analysis, His6-tagged c-FLIP-transfected cells in 10-cm dishes were lysed in 1 ml of 50 mm NaH2PO4, pH 8.0, containing 300 mm NaCl, 10 mm imidazole, 0.05% Tween 20, and a protease and phosphatase inhibitor mixture at 4 C for 45 min on a wheel rotor and complemented with sonication (6 pulses) on ice. The lysates were centrifuged at 10,000 for 15 min, then the resulting supernatants were incubated with Ni-NTA-agarose beads at 4 C for 2 h. After a brief centrifugation, the beads were washed three times in washing buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 20 mm imidazole, 0.05% Tween 20) and the bound proteins were released in elution buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 250 mm imidazole, 0.05% Tween 20) and combined with Laemmli sample Azilsartan (TAK-536) Azilsartan (TAK-536) buffer; proteins were separated by (11%) SDS-PAGE before being digested with trypsin for mass spectrometry analysis. For direct immunoblot analysis using cell lysates, cells were lysed with RIPA DLK buffer (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100) containing a protease and phosphatase inhibitor mixture at 4 C for 30 min on a wheel rotor. The lysates were centrifuged at 10,000 for 15 min. FLIP levels were quantified by densitometry using ImageJ Azilsartan (TAK-536) software. For sequential probing with multiple antibodies, the blots were stripped using 0.1 m glycine, pH 2.5, with 0.1% Tween at 37 C for 30C60 min and washed with Tris-buffered saline containing 0.1% Tween 20 (TBST20). Measurement of Cellular Reactive Oxygen Species After treatment with menadione, cells were washed twice with phosphate-buffered saline (PBS) and combined with 10 m H2DCFDA in PBS answer. Cultures were incubated at 37 C under 5% CO2 for 30 min. Any unbound dye was removed by washing with PBS, then the cells were trypsinized and cell pellets were washed and resuspended in PBS. Fluorescence (excitation 485 nm, emission 530 nm) was measured by fluorescence-activated cell scanning (FACS; BD Biosciences). FLIP mRNA Analysis Following the treatment of.