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. 2023 Jun 17;26(7):107159. doi: 10.1016/j.isci.2023.107159

Figure 1.

Figure 1

BiFCPL enables the labeling of proteins at mitochondria-ER contacts

(A) Designing principle of the method.

(B) Fluorescent images of biotinylated proteins. U2OS or MERC cells were transiently transfected with GNAP2. After biotin labeling, the cells were fixed and subjected to fluorescence labeling. Alexa 555-conjugated streptavidin was used to detect biotin-labeled proteins. Alexa 647-conjugated Tom20 antibody was used to label mitochondria. Scale bar, 5 μm. See also Figure S1. Contact, green; streptavidin, magenta; blue, mitochondria.

(C) Western blotting analysis of the enriched proteins. U2OS or MERC cells were transiently transfected with GNAP2, NLS-GNAP2, or CS-GNAP2. The cells were incubated with or without biotin-phenol for biotin labeling. The cells were then harvested, and biotin-labeled proteins were enriched by streptavidin magnet beads, separated by SDS-PAGE and detected with HRP-conjugated streptavidin (SA-HRP).

(D) Western blotting analysis of the enriched proteins. Enriched proteins were subjected to SDS-PAGE and immunoblotted with antibodies against MFN2, RMDN3, GFP, and ACTB.