Figure 5.
Figure 5. High-affinity anti-TfR colocalizes with lysosomes. (A and D) bEND.3 cells were incubated with 600 nM control IgG, 20 nM anti-TfRA/control (Ctr), or 600 nM anti-TfRD/control for 20, 60, and 120 min. The cells were fixed and immunostained for EEA1 and for the incubated antibodies with Alexa Fluor 594 anti–human IgG (red) and quantified for relative colocalized percent areas. (A) Representative confocal images of 20- and 120-min antibody incubations. (D) Quantification of colocalized percent area detected with EEA1 and anti–human IgG normalized to total percent area detected by anti-EEA1. (B) Representative images of anti-Lamp1 and control IgG, anti-TfRA/control, or anti-TfRD/control (at their respective IC50 concentrations) at 20 and 120 min. (E) Quantification for colocalized percent area for LAMP1 and each incubated antibody at the indicated time points normalized to LAMP1 total percent area. (C) bEND.3 cells were incubated with Alexa Fluor 488–labeled human holo-Tf (hu-Tf) at 400 nM for 20, 60, and 120 min and subsequently fixed and stained for EEA1. (F) Quantification of colocalized percent area detected with anti-EEA1 and Tf-488 and normalized to total percent area detected by EEA1. All bispecific antibody and Tf incubations were performed at 37°C. Data shown are the means ± SEM of n = 3 independent experimental repeats; within each experiment four fields were quantified from 100× confocal images of single optical z-planes. P-values were obtained by Student’s t test versus control IgG (D and E) or versus 20-min time point (F): *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (G and H) In vivo imaging of cortical blood vessels labeled with AngioSense vascular dye (red) 1 and 20 h after i.v. injections with 10 mg/kg pHrodo–anti-TfRA/control, pHrodo–anti-TfRD/control, or pHrodo–control IgG. Punctate labeling of acidic compartments (white) is shown. Representative images from two different mice are shown for each antibody condition; n = 2 mice imaged per condition, with three to four imaging fields per mouse. Bars: (A–C and H) 5 µm; (G) 10 µm.

High-affinity anti-TfR colocalizes with lysosomes. (A and D) bEND.3 cells were incubated with 600 nM control IgG, 20 nM anti-TfRA/control (Ctr), or 600 nM anti-TfRD/control for 20, 60, and 120 min. The cells were fixed and immunostained for EEA1 and for the incubated antibodies with Alexa Fluor 594 anti–human IgG (red) and quantified for relative colocalized percent areas. (A) Representative confocal images of 20- and 120-min antibody incubations. (D) Quantification of colocalized percent area detected with EEA1 and anti–human IgG normalized to total percent area detected by anti-EEA1. (B) Representative images of anti-Lamp1 and control IgG, anti-TfRA/control, or anti-TfRD/control (at their respective IC50 concentrations) at 20 and 120 min. (E) Quantification for colocalized percent area for LAMP1 and each incubated antibody at the indicated time points normalized to LAMP1 total percent area. (C) bEND.3 cells were incubated with Alexa Fluor 488–labeled human holo-Tf (hu-Tf) at 400 nM for 20, 60, and 120 min and subsequently fixed and stained for EEA1. (F) Quantification of colocalized percent area detected with anti-EEA1 and Tf-488 and normalized to total percent area detected by EEA1. All bispecific antibody and Tf incubations were performed at 37°C. Data shown are the means ± SEM of n = 3 independent experimental repeats; within each experiment four fields were quantified from 100× confocal images of single optical z-planes. P-values were obtained by Student’s t test versus control IgG (D and E) or versus 20-min time point (F): *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (G and H) In vivo imaging of cortical blood vessels labeled with AngioSense vascular dye (red) 1 and 20 h after i.v. injections with 10 mg/kg pHrodo–anti-TfRA/control, pHrodo–anti-TfRD/control, or pHrodo–control IgG. Punctate labeling of acidic compartments (white) is shown. Representative images from two different mice are shown for each antibody condition; n = 2 mice imaged per condition, with three to four imaging fields per mouse. Bars: (A–C and H) 5 µm; (G) 10 µm.

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