Figure 2.
Figure 2. Capturing of IFNAR2 into micropatterns. (a) IFNAR2 (blue) fused to EGFP (green) and the HaloTag (dark green) was transiently transfected into HeLa cells and cultured on a micropatterned support. Ligand binding was probed by incubating 10 nM AT655IFNα2 (red–orange). (b) Fluorescence intensity within a single HeLa cell expressing HaloTag-EGFP-IFNAR2 (green channel) after incubation of AT655IFNα2 (red channel). The overlay of both channels is shown in the right image. Dotted lines indicate the boundaries of the cells. Bars, 10 µm. (c–e) Reversible ligand binding quantitatively probed by single-molecule imaging of the low-affinity mutant DY647IFNα2 M148A-NLYY. (c) Superimposition of 200 consecutive frames in the presence of 0.5 nM DY647IFNα2 M148A-NLYY before (left) and after (right) chasing with unlabeled IFNα2-α8tail-YNS. Dotted lines indicate the analyzed line pattern. Bar, 5 µm. (d) Dwell time distribution obtained from single-molecule localization experiments in micropatterns (20,870 binding events from the cell shown in c, representative for three cells analyzed). Only molecules localized for >10 consecutive frames (320 ms) were evaluated. (e) Displacement kinetics in micropatterns upon chasing with unlabeled IFNα2-α8tail-YNS (injection marked with an arrow) and fit of the curve (representative of three cells analyzed).

Capturing of IFNAR2 into micropatterns. (a) IFNAR2 (blue) fused to EGFP (green) and the HaloTag (dark green) was transiently transfected into HeLa cells and cultured on a micropatterned support. Ligand binding was probed by incubating 10 nM AT655IFNα2 (red–orange). (b) Fluorescence intensity within a single HeLa cell expressing HaloTag-EGFP-IFNAR2 (green channel) after incubation of AT655IFNα2 (red channel). The overlay of both channels is shown in the right image. Dotted lines indicate the boundaries of the cells. Bars, 10 µm. (c–e) Reversible ligand binding quantitatively probed by single-molecule imaging of the low-affinity mutant DY647IFNα2 M148A-NLYY. (c) Superimposition of 200 consecutive frames in the presence of 0.5 nM DY647IFNα2 M148A-NLYY before (left) and after (right) chasing with unlabeled IFNα2-α8tail-YNS. Dotted lines indicate the analyzed line pattern. Bar, 5 µm. (d) Dwell time distribution obtained from single-molecule localization experiments in micropatterns (20,870 binding events from the cell shown in c, representative for three cells analyzed). Only molecules localized for >10 consecutive frames (320 ms) were evaluated. (e) Displacement kinetics in micropatterns upon chasing with unlabeled IFNα2-α8tail-YNS (injection marked with an arrow) and fit of the curve (representative of three cells analyzed).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal