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. 2014 Nov 10;207(3):407–418. doi: 10.1083/jcb.201406032

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© 2014 Löchte et al.

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Figure 5. — Recruitment and activation of JAKs. (a) Binding of tyk2 to micropatterned IFNAR1. HeLa cells cotransfected with HaloTag-IFNAR1 and tyk2-EGFP (green channel) were cultured on a micropatterned support and stained with ^AT655IFNα2-YNS (red channel). Cell boundaries are indicated by a dotted line. Bars, 20 µm. (b) Sectional profiles of tyk2 and IFNAR1 fluorescence intensity in the respective channels of a (representative of five cells analyzed). Integrated line profiles were scaled to similar amplitudes and overlaid at an arbitrary ordinate scale. a.u., arbitrary unit. (c) FRAP of tyk2 within the pattern. The bleached area is indicated by a dotted circle. (right) Projected profile across the bleached area as indicated in the inset (representative of four cells analyzed). rel., relative. Bar, 10 µm. (d) FRAP curves obtained in different ROIs within c color coded as indicated in the inset (representative of four cells analyzed). (e) Formation of a functional ternary signaling complex probed by immunostaining of pJak1. HeLa cells were transfected with HaloTag-TagRFP-IFNAR1 (yellow channel) and SNAP-IFNAR2. After addition of ^AT655IFNα2 (red channel), cells were fixed and stained via an anti-pJak1 antibody (green channel). Cell boundaries are indicated by a dotted line. Bars, 10 µm.