Dataset Information

GST-SH2 domain of 3BP2, pull down, curdlan-stimulated BMDC

ABSTRACT: Interacting molecules with GST-fusion protein of the wild-type SH2 domain of 3BP2 (WT) or the mutant form (R486K) were precipitated usng GSH beads and identified by DIA proteome analysis.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Kazuyasu Chihara

PROVIDER: PXD054171 | JPOST Repository | Fri Nov 15 00:00:00 GMT 2024

REPOSITORIES: jPOST

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Publications

Adaptor protein 3BP2 regulates gene expression in addition to the ubiquitination and proteolytic activity of MALT1 in dectin-1-stimulated cells.

Tsubokawa Ayumi A Chihara Kazuyasu K Chihara Yuri Y Takeuchi Kenji K Fujieda Shigeharu S Sada Kiyonao K

The Journal of biological chemistry 20241113 12

Dectin-1, a C-type lectin, plays important roles in the induction of antifungal immunity. Caspase recruitment domain-containing protein 9 (CARD9) is essential for the dectin-1-induced production of cytokines through the activation of NF-κB. However, the molecular mechanisms underlying the dectin-1-mediated activation of CARD9 have not been fully elucidated. Recently, we reported that the adaptor protein SH3 domain-binding protein 2 (3BP2) is required for the dectin-1-induced production of cytoki ...[more]

PMID: 39542253

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Project description:RNA pull-down assay. For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified. RNA purification and amplification: RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual. Microarray analysis: The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.

			Action	DRS
	KK2601-1_WT.mzML	Mzml
	KK2601-1_WT.raw	Raw
	KK2601-2_RK.mzML	Mzml
	KK2601-2_RK.raw	Raw
	KK2601.auto.pipeline	Other