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APEX2-based proximity labelling of Staphylococcus aureus receptors on human lung microvascular endothelial cells

ABSTRACT: Staphylococcus aureus bacteria were decorated with modified ascorbate peroxidase APEX2-YFP-CWT via the cell wall targeting (CWT) domain of lysostaphin. Cytosol of transgenic HeLa cells expressing APEX2-YFP-CWT was generated by ultracentrifugation and living bacteria were incubated in the cytosol. The bacteria were stringently washed and added to either untreated human lung microvascular endothelial cells (HuLEC) or to cells pre-treated with bacterial sphingomyelinase (β-toxin), ionomycin, or amitriptyline. After 15 min, H2O2 was added for 1 min, then the biotinylation reaction was stopped by addition Stop Solution (containing sodium azide, sodium ascorbate, and Trolox in PBS). The cells then were scraped off the substratum, centrifuged, and lysed in RIPA buffer. To remove excess biotin phenol, lysates were transferred into a 3 kDa cutoff Amicon concentrator and 5 ml RIPA buffer was added. Solution was concentrated by centrifugation to 500-1000 µL. This step was repeated once. The lysates then were incubated overnight with magnetic streptavidin beads, followed by extensive washing and eventually biotinylated proteins were eluted by addition of Laemmli buffer containing an excess of biotin.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Staphylococcus Aureus

TISSUE(S): Cell Culture, Endothelial Cell

DISEASE(S): Opportunistic Bacterial Infectious Disease

SUBMITTER: Martin Fraunholz

LAB HEAD: Martin J Fraunholz

PROVIDER: PXD051937 | Pride | 2025-03-13

REPOSITORIES: Pride

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			Action	DRS
	2023-02-10_Fusion_36237.raw	Raw
	2023-02-10_Fusion_36241.raw	Raw
	2023-02-10_Fusion_36245.raw	Raw
	2023-02-10_Fusion_36249.raw	Raw
	2023-06-07_Fusion_37872.raw	Raw

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