Dataset Information

Immunnoprecipitation followed by PRM to detect endogenous interaction between VGLL3 and DDX5

ABSTRACT: For immunoprecipitation to detect the endogenous interaction between VGLL3 and DDX5, cardiac myofibroblasts were fixed with 0.1% PFA and lysed in IP buffer [20 mM HEPESNaOH (pH 7.5)/1 mM EGTA/1 mM MgCl2/150 mM NaCl /5% glycerol/1% NP40] containing protease inhibitors (1:100) and phosphatase inhibitors (1:100) at 4 °C for 20 min. The supernatants of cell lysates were incubated for 2 h at 4 °C with SureBeads Protein G (Bio-Rad) coupled with the custom-made rabbit polyclonal anti-VGLL3 antibody, raised against the amino acid sequence of mouse VGLL3 from position 307 to 325. The beads were then washed four times with IP buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested with 200 ng of Trypsin/Lysyl Endopeptidase mixture, and the digested peptides were reduced, alkylated, acidified, desalted, and dissolved in 0.1% trifluoroacetic acid and 3% ACN. To quantify VGLL3 and DDX5, over four peptides of VGLL3 and DDX5 were measured by PRM, an MS/MS-based targeted quantification method using high-resolution MS. Targeted MS/MS scans were acquired by a time-scheduled inclusion list at a resolution of 70,000, an AGC target of 2e5, an isolation window of 2.0 m/z, a maximum injection time of 1 s, and a normalised collision energy of 27. Time alignment and relative quantification of transitions were performed using Skyline software

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD039093 | JPOST Repository | Sat Dec 24 00:00:00 GMT 2022

REPOSITORIES: jPOST

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Publications

VGLL3 is a mechanosensitive protein that promotes cardiac fibrosis through liquid-liquid phase separation.

Horii Yuma Y Matsuda Shoichi S Toyota Chikashi C Morinaga Takumi T Nakaya Takeo T Tsuchiya Soken S Ohmuraya Masaki M Hironaka Takanori T Yoshiki Ryo R Kasai Kotaro K Yamauchi Yuto Y Takizawa Noburo N Nagasaka Akiomi A Tanaka Akira A Kosako Hidetaka H Nakaya Michio M

Nature communications 20230208 1

Myofibroblasts cause tissue fibrosis by producing extracellular matrix proteins, such as collagens. Humoral factors like TGF-β, and matrix stiffness are important for collagen production by myofibroblasts. However, the molecular mechanisms regulating their ability to produce collagen remain poorly characterised. Here, we show that vestigial-like family member 3 (VGLL3) is specifically expressed in myofibroblasts from mouse and human fibrotic hearts and promotes collagen production. Further, subs ...[more]

PMID: 36754961

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			Action	DRS
	1_siCont_IgG_PRM1.raw	Raw
	1_siCont_IgG_PRM2.raw	Raw
	1_siCont_IgG_PRM3.raw	Raw
	200925_Nakaya_IP_PRM_4th_n3.csv	Csv
	2_siVGLL3_IgG_PRM1.raw	Raw