Dataset Information

Interactome of human BAG3 in A7r5 rat smooth muscle cells

ABSTRACT: The interactome of humanBAG3 phosphosite mutant variants in rat smooth muscle cells

INSTRUMENT(S): maXis

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human)

TISSUE(S): Cell Culture, Aortic Smooth Muscle Cell

DISEASE(S): Disease Free

SUBMITTER: Henrique Baeta

LAB HEAD: Pitter Huesgen

PROVIDER: PXD048551 | Pride | 2024-08-15

REPOSITORIES: Pride

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			Action	DRS
	2202024_A_HB_BAG3_IP_BAG_2_BA3_1_4110.d.zip	Other
	2202024_A_HB_BAG3_IP_BAG_2_BA3_1_4144.d.zip	Other
	2202024_A_HB_BAG3_IP_BAG_2_BA3_1_4178.d.zip	Other
	2202024_A_HB_BAG3_IP_BAG_3_BA11_1_4138.d.zip	Other
	2202024_A_HB_BAG3_IP_BAG_3_BA11_1_4172.d.zip	Other

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Project description:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.

Dataset Information

Interactome of human BAG3 in A7r5 rat smooth muscle cells

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