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1.
Figure 4

Figure 4. Detection of TfR1-PCP Using Biotin Labeling. From: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling.

TRVb/TfR1-PCP or TRVb cells were incubated with or without biotin-CoA (1) in the presence and absence of Sfp. Cell lysates were prepared for electrophoresis under both nonreducing (A) and reducing (B) conditions. After transfer to a PVDF membrane, labeling with biotin was probed using streptavidin-HRP.

Jun Yin, et al. Chem Biol. ;12(9):999-1006.
2.
Figure 1

Figure 1. Model for Transferrin-Mediated Iron Uptake. From: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling.

Transferrin receptor 1 (green) is drawn with C terminus fused to the PCP domain (blue), and transferrin ligand is shown as red triangles with iron (gray balls) bound. For fluorescence imaging, transferrin receptor 1 was labeled with Alexa 488 (green oval) attached to the PCP, and transferrin was labeled with Alexa 568 (red circle).

Jun Yin, et al. Chem Biol. ;12(9):999-1006.
3.
Figure 7

Figure 7. Time Course of FRET Redistribution in TRVb Cells during Transferrin Uptake. From: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling.

TRVb cells were transfected with TfR1-PCP, labeled with CoAAlexa Fluor 488 (2), and incubated with Tf-Alexa 568. Images were calculated from data collected at incubation times of 2 min (A), 5 min (B), and 15 minutes (C). Scale bar = 10 μm.

Jun Yin, et al. Chem Biol. ;12(9):999-1006.
4.
Figure 5

Figure 5. Transferrin Uptake Pathway. From: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling.

Cells were incubated with Alexa Fluor 568-labeled transferrin, fixed at 1, 10, and 30 min after incubation, and imaged using confocal microscopy. Stacks of optical slices at 0.25 μm per step were processed by MetaMorph using 2D no neighbors deconvolution before 3D reconstruction. Top: TRVb1 cells (100% confluency). Bottom: TRVb cells transfected with TfR1-PCP prior to the addition of transferrin (80%–90% confluency); note that nontransfected cells are not visible. Scale bar = 10 μm.

Jun Yin, et al. Chem Biol. ;12(9):999-1006.
5.
Figure 2

Figure 2. Sfp-Catalyzed Labeling of TfR1-PCP Fusion on the Surface of the Cell. From: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling.

Biotin and Alexa Fluor 488 are conjugated to CoA (1 and 2), and Sfp catalyzes the specific modification of a conserved serine residue on the PCP (red ribbon) by the phosphopantetheinyl group attached to the small molecule labels. PCP is shown fused to the C terminus (red) of the extracellular domain (blue) of TfR1 with its N terminus and transmembrane domain in green. Transferrin (gray) binds to TfR1 with a 2-fold symmetry. The structure of transferrin-TfR1 complex was adapted from [23].

Jun Yin, et al. Chem Biol. ;12(9):999-1006.
6.
Figure 3

Figure 3. Functional Characterization of TfR1-PCP Fusion. From: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling.

(A) Cell-associated 55Fe was measured in uptake assays upon incubation of TRVb, TRVb1, and TRVb/TfR1-PCP cells with 40 nM 55Fetransferrin, as described in Experimental Procedures. Also shown is a Western blot for TRVb (1), TRVb1 (2), and TRVb/TfR1-PCP (3) cell lysates probed with mouse anti-TfR1 antibody.
(B) Surface 125I-transferrin binding was measured for TRVb1 and TRVb/TfR1-PCP cells incubated on ice with 0.05–500 nM 125I-transferrin in the presence or absence of excess unlabeled transferrin. Specific cell surface binding was taken as the difference between these measured values, and the data were normalized to maximal values determined at 500 nM 125I-transferrin for each cell line. Binding curves were fit assuming a 1:1 binding model.

Jun Yin, et al. Chem Biol. ;12(9):999-1006.
7.
Figure 6

Figure 6. FRET Analysis of Transferrin-TfR1 Interactions in TRVb Cells Transfected with TfR1-PCP. From: Single-Cell FRET Imaging of Transferrin Receptor Trafficking Dynamics by Sfp-Catalyzed, Site-Specific Protein Labeling.

TfR1 was fluorescently labeled with CoAAlexa Fluor 488 (2), and cells were then incubated with Tf-Alexa 568 for 2 min. Shown are images of TfR1-PCP/CoA-Alexa Fluor 488 (2) before (A) and after (B), and Tf-Alexa 568 before (C) and after (D), photobleaching of the acceptor fluorophore Alexa Fluor 568. (E) Shows the calculated relative intensity of FRET between donor and acceptor fluorophores, and (F) represents the overlay of (B) (red), (C) (green), and (E) (blue). (G) Linescan results by MetaMorph. Fluorescence intensities of the donor (red) and the acceptor (green) were measured along the line drawn in (F). Calculated FRET intensity along the same line (blue) was present only where both donor and acceptor fluorophores were present. Scale bar = 10 μm.

Jun Yin, et al. Chem Biol. ;12(9):999-1006.

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