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Granzyme A can be modified by 4-OI

ABSTRACT: HEK293T cells that transfected with plasmid encoding human FLAG-Granzyme A (Sino Biological, HG13325-NF) using lipofectamine 3000 for 24 h were treated with 4-OI (0.5 mM) for 6h.Following treatment, cells were lysed in lysis buffer and immunoprecipitated with anti-FLAG antibody and protein A/G agarose beads. Next the beads were washed three times with 1ml lysis buffer after centrifuging at 400g for 2 min. The immune complexes were eluted by addition of 40 ml lysis buffer, dissolved in 1x SDS sample buffer and boiled for 5 min. The samples were separate by SDS-PAGE and subsequently probed with Coomassie blue. The corresponding bands for FLAG-Granzyme A were excised from the gel and subjected to in-gel digest with trypsin. Briefly, the gel slices were cut into smaller pieces before reduction with dithiothreitol (10 mM) and then dehydrated in 100% acetonitrile. Gel slices were digested with trypsin overnight at 37℃. Peptides were eluted from the gel pieces following protease digest and dried down completely in a vacuum centrifuge. Samples were analyzed in a Q Exactive TM Plus (Thermo Fisher) coupled online to UPLC after the nanospray ionization source. MS data was analyzed with Proteome Discoverer 1.3 and tandem mass spectra were searched against the UniProt database. Trypsin/P is designated as a cleavage enzyme that allows up to two deletion cleavage. Precursor mass tolerance was set to 10 ppm, while fragments were detected with a tolerance of 0.02 Da.

ORGANISM(S): Homo Sapiens

SUBMITTER: Wenbin Gong

PROVIDER: PXD047364 | iProX | Wed Nov 29 00:00:00 GMT 2023

REPOSITORIES: iProX

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

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Granzyme A can be modified by 4-OI

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