MassIVE MSV000098126

Partial Public PXD064891

PERK protein kinase facilitates keratinocyte collective cell migration by engagement with cell adhesion molecules independent of its kinase activity

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Description

Proximity labeling experiments (BioID) were performed on Human NTERT keratinocytes depleted of PERK (EIF2AK3, PERK-KO) re-expressing a WT PERK containing a C-terminal UltraID tag. PERK-KO and PERKUltraID cells were treated with differentiation medium containing 50 uM of exogenous Biotin for 6 h, washed three times with ice-cold PBS and harvested on ice with a modified RIPA buffer solution (25mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA, 1x HALT protease/phosphatase inhibitor cocktail). Samples were then sonicated and incubated at 4C with rotation for 30 minutes, followed by centrifugation step to pellet cell debris. Total protein concentration was measured, and the samples were normalized to 1 mg/ml to a final volume of 1 ml. Normalized samples were incubated with 40uL of trypsin-resistant streptavidin beads with rotation overnight at 4C. The next day, the beads were collected by magnetic separation and washed three times with an IP wash solution (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100), followed by two washes with PBS. Finally, the beads were resuspended with 100 ul of PBS and submitted on ice for preparation and mass spectrometry analysis by the Indiana University School of Medicine Center for Proteome Analysis. [doi:10.25345/C5NZ8129R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: BioID ; MS ; proteomics ; NTERT ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Amber L. Mosley, Indiana University School of Medicine, USA
Dan F. Spandau, Indiana University School of Medicine, United States
Ronald C. Wek, Indiana University School of Medicine, USA
Submitting User: edoud

Publications

Diaz MB, Staschke KA, Baucum AJ, Spandau DF, Wek RC.
PERK protein kinase facilitates keratinocyte collective cell migration by engagement with cell adhesion molecules independent of its kinase activity.
Mol Biol Cell. Epub 2025 Sep 24.

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