The ten most intense ions were sequentially isolated and fragmented by higher energy C\trap dissociation (HCD) (Olsen em et al /em , 2007)

The ten most intense ions were sequentially isolated and fragmented by higher energy C\trap dissociation (HCD) (Olsen em et al /em , 2007). Bioscience, Heidelberg, Germany), anti\phospho\RIPK1 (#65746S, Cell Signaling), anti\MLKL (#14993S, Cell Signaling), anti\phospho\MLKL (S358) (#91689S, Cell Signaling), anti\\Actin (#A5441, Sigma\Aldrich), anti\GAPDH (#5G4\6C5, HyTest, Ltd., Turku, Finland), anti\HA (F\7) (#sc\7392x, Santa Cruz Biotechnology), anti\Strep\tag II (#ab76949, Abcam), anti\His\tag (#sc\53073, Santa Cruz Biotechnology), anti\USP22 (#NBP1\49644, Novus Biological, Centennial, Colorado, USA), anti\USP22 (#ab195298, Abcam), anti\Vinculin (#V9131\100UL, Merck), anti\Histone H2B (#07\371, Merck), anti\Ubiquityl\Histone H2B (#05\1312, Merck), anti\Cas9 (7A9) (#MAC133, Merck), anti\IB (#9242S, Cell Signaling), anti\phospho\IB (#9246L, Cell Signaling), anti\caspase\8 (#9746S, Cell Signaling), anti\caspase\8 (#ADI\AAM\118\E, Enzo Life Sciences), anti\TRADD (#ab110644, Abcam), anti\FADD (#610400, BD Bioscience), and anti\TNFR1 (#3736S, Cell Signaling). For detection, horseradish peroxidase (HRP)\labeled goat anti\mouse IgG and goat anti\rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies and detected with enhanced chemiluminescence (Amersham Bioscience, Freiburg, Germany). Primary antibodies were diluted in 1:1,000 in PBS with 0.2% Tween 20 (PBS\T) containing 2% bovine serum albumin (BSA), and secondary antibodies were diluted 1:10,000 in PBS\T with 5% milk powder. Representative blots of at least two independent experiments are shown. Multiple loading controls indicate independent Western blot membranes used to obtain clearer signals due Razaxaban to membrane stripping. Determination of cell death The indicated cell lines were seeded in appropriate densities in sterile 96\well plates (Greiner Bio\One, Kremsmnster, ?sterreich) one day prior to stimulation with the indicated concentrations of zVAD.fmk, BV6, and human recombinant TNF. Cell death was assessed by fluorescence\based microscopic quantification of the fraction of PI\positive cells, compared to total cells, using Hoechst 33342 and PI double staining (Sigma\Aldrich). Imaging and quantification were performed using the ImageXpress Micro XLS Widefield High\Content Analysis System and MetaXpress Razaxaban Software according to the manufacturer’s instructions (Molecular Devices Sunnyvale, CA, USA). RNA interference Transient genetic silencing of USP22 was performed by reverse transfection of cells with 20?nM Silencer Select siRNAs (Life Technologies, Inc.) using Lipofectamine RNAiMax reagent (Life Technologies, Inc.) and Opti\MEM medium (Life Technologies, Inc.). The following siRNAs were used in this study: human siUSP22 (#1: s230743 #2: s230744) mouse siUSP22 (#1: s103730, #2: s103729) and non\targeting control siRNA (#4390843). Knockdown efficiency was confirmed by Western blot analysis. Generation of USP22 and RIPK3 CRISPR/Cas9 KO cells To generate CRISPR/Cas9 control (targeting eGFP), human and mouse USP22?KO or RIPK3 KO cells, guide RNAs for control (Addgene plasmid #51763, # 51762 and # 51760), USP22 human (#1:GCCATTGATCTGATGTACGG, #2: CCTCGAACTGCACCATAGGT and #3: ACCTGGTGTGGACCCACGCG), USP22 mouse (#1:CCGTACATCAGGTCGATGGC, #2:ACCCGTAAAGATCTGGTCAA, #3: CAGGTTGATCAGTCCACGCA GAL and #4:CTGTGAGATGCAGAGCCCCA) or RIPK3 (GTTTGTTAACGTAAACCGGA) were cloned into plentiCRISPRv2 (Addgene plasmid #52961) (Sanjana (de)ubiquitination immunoprecipitations For (de)ubiquitination immunoprecipitations under denaturing conditions, cells were either lysed in NP\40 lysis buffer, supplemented with 1% SDS, or in RIPA lysis buffer (see above), supplemented with 2% SDS and 25?mM?N\ethylmaleimide (NEM), before being sonificated (three cycles, Razaxaban 10?s burst, amplitude 25, 30?s rest), and boiled at 95C for 10?min. Lysates were incubated for 30?min at 4C. NP\40 buffer lysates were then diluted with regular lysis buffer (1:10), whereas RIPA lysates were diluted 1:10 in Ni\NTA lysis buffer (6?M Guanidium HCl, 0.1?M NaH2PO4/Na2HPO4, 100?mM Tris, pH 8). At least 1.5?mg of protein lysate was incubated with either Anti\HA Magnetic Beads (#8883, Thermo Fisher Scientific) or HisPur? Ni\NTA beads (#88832, Thermo Fisher Scientific) and rotated overnight at 4C. Beads were pre\washed twice with either NP\40 lysis buffer or Ni\NTA lysis buffer, respectively. The next day, Anti\HA Magnetic Beads were washed five times with NP\40 buffer and boiled for 5?min at 95C in 2x Laemmli loading buffer. Ni\NTA beads were washed twice with Ni\NTA washing buffer 1 (6?M Guanidium HCl, 0.1?M NaH2PO4/Na2HPO4, 10?mM Tris, 0.05% Triton X\100, pH 8), 2 (8?M urea, 0.1?M NaH2PO4/Na2HPO4, 10?mM Tris, 0.05% Triton X\100, pH 8), and 3 (8?M urea, 0.1?M NaH2PO4/Na2HPO4, 10?mM TrisCHCL, 0.05% Triton X\100, pH 6.3) before being washed once in PBS. Beads were then centrifuged at 855 for 1? min before being eluted by constantly vortexing the beads for 30?min with Ni\NTA elution buffer (3x Laemmli loading buffer, 200?mM Imidazole).

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