CircITGB6-pulled down proteins in A549 cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell, Cell Culture
SUBMITTER: 
ke li
LAB HEAD: Ke Li
PROVIDER: PXD043341 | Pride | 2023-10-13
REPOSITORIES: Pride
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2019-10-15 | GSE134103 | GEO
Project description:Ten 15 cm2 plates of HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were lysed with TAP lysis buffer (10% (v/v) glycerol, 50 mM HEPES-KOH pH 7.5, 100 mM KCl, 2 mM EDTA, 0.1% (v/v) NP-40, 10 mM NaF, 0.25 mM Na3VO4, 50 mM β-glyerolphosphate, 2 mM DTT, and 1X protease inhibitor cocktail (Sigma)) and cleared supernatants were bound to IgG-Sepharose beads (GE Amersham) and then washed in lysis buffer and TEV buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM DTT, and 1X protease inhibitor cocktail). Protein complexes were cleaved off the beads by TEV protease (Sigma) and incubated with calmodulin-Sepharose beads (GE Amersham) in calmodulin binding buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 1 mM Mg acetate, 1 mM imidazole, 0.1% (v/v) NP-40, 6 mM CaCl2, 10 mM 2-mercaptoethanol) and then washed in calmodulin rinse buffer (50 mM Ammonium bicarbonate pH 8.0, 75 mM NaCl, 1 mM Mg Acetate, 1 mM Imidazole, 2 mM CaCl2) before eluted with SDS sample buffer, subjected to SDS-PAGE, and stained with GelCode Blue stain (Thermo Fisher Scientific) before protein identification by LC-MS/MS.
2020-09-01 | PXD020231 | Pride
Project description:Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)
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Project description:We generated a HEK293 cell line expressing ZZ-TEV-FLAG-bait protein by Lipofectamine 3000 transfection. The expression level of bait protein is controlled to endogenous level by addition of 2 ng/mL doxycycline. Note the combined treatment of Torin1 and CQ was applied for STX17. The tandem affinity purification of tagged bait protein was performed in a physiology buffer condition [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Igepal CA-630, 1 mM EDTA, protease inhibitor cocktail (Roche)]. The eluted protein mixtures were subjected to SDS/PAGE and further analyzed by mass spectrometry.
2023-09-18 | PXD043048 | Pride
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
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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2022-12-07 | PXD023141 | Pride
Project description:0.3g of flowers were ground in liquid nitrogen. The powder was extracted for 30 min [H2] in 1.5 ml of lysis buffer (50mM Tris HCl pH 8.0, 50mM NaCl, 1% Triton x-100 and 1 x cOmplete™ EDTA-free protease inhibitor (Roche)). After removal of cell debris by centrifugation (2 times 10 min, 16000 x g, 4 °C) the cleared supernatants were incubated for 30 min with GFP or FLAG antibodies coupled to magnetic microbeads (μMACS GFP and DYKDDDDK isolation Kits, Miltenyi). Beads were loaded on magnetized MACS separation columns equilibrated with lysis buffer and washed 4 times with 300 µl washing buffer (50mM Tris HCL pH 7.5, 25 or 50 mM NaCl, 0,1 % Triton). Samples were eluted in 50 µl of pre-warmed elution buffer (Milteny). Control IPS were performed in Col-O using either GFP or FLAG antibodies.
2020-04-15 | PXD014447 | Pride
Project description:For modified iCLIP experiment, 4SU was used for crosslinking as described in published protocol (citation is bellow) and the RNase conditions were optimised to ensure efficient RNase I-dependent fragmentation. In detail, HEK293T cells were grown on 10 cm 2 dishes, incubated for 8 h with 100 M 4SU and crosslinked with 2x 400mJ/cm 2 365nm UV light. Protein A Dynabeads were used for immunoprecipitations (IP). 80 l of beads were washed in iCLIP lysis buffer (50mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). For the preparation of the cell lysate, 2 million cells were lysed in 1 ml of iCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) buffer, and the remaining cell pellet was dissolved in 50 L MSB lysis buffer (as above). After the pellet had dissolved, the mixture was diluted with CLIP lysis buffer to 1000 l and an additional centrifugation was performed. Lysates were pooled (2ml total volume) and incubated with 4 U/ml of RNase I and 2 l antiRNase (1/1000, AM2690, Thermo Fisher) at 37C for 3 min, and centrifuged. We took care to prepare the initial dilution of RNase in water, since we found that RNase I gradually loses its activity when diluted in the lysis buffer. 1.5 ml of the supernatant was then added to the beads and incubated at 4C for 4 h. The rest of the protocol was identical to the published protocol (see bellow). Huppertz I, Attig J, D'Ambrogio A, Easton LE, Sibley CR, Sugimoto Y, Tajnik M, Knig J, Ule J: iCLIP: protein-RNA interactions at nucleotide resolution. Methods 2014, 65:274-287.
2016-11-24 | E-MTAB-5027 | biostudies-arrayexpress
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