Figure 7. Analysis of conformational changes in p75^NTR intracellular domains by anisotropy microscopy.

(A–D) Steady-state anisotropy in transfected cells. Examples of areas used for anisotropy measurements are boxed and shown as high magnification insets. Monomeric EGFP (EGFP1, high anisotropy, low FRET) and a concatenated EGFP trimer (EGFP3, low anisotropy, high FRET) were used as controls. The calibration bar of the look-up table is shown below.

(E) Steady-state anisotropy of wild type and C257A p75^NTR-EGFP in COS-7 cells. The anisotropy value of EGFP1 was arbitrarily set to zero and used as baseline for the histogram. Bars show average ± SD (n=8–11 cells for EGFP and 22–30 cells for p75^NTR).

(F) Representative examples of anisotropy traces after addition of NGF or medium in cells expressing wild type or C257A p75^NTR-EGFP. A peak in anisotropy was observed after NGF addition in all cells expressing wild type p75^NTR that were examined (n=30) regardless of their initial baseline anisotropy level.

(G) Anisotropy change after addition of NGF or medium (control). The difference in anisotropy before and after addition of NGF (i.e. peak minus baseline value) or medium was calculated for wild type and C257A p75^NTR-EGFP. Results are expressed as average ± SD (n=15 to 17 cells examined). *, p<0.0001 vs. C257A.