Fig. 4.

Cholesterol submicrometric domain abundance and theta* binding to cholesterol depend on temperature. A Confocal imaging. RBCs were labeled with theta*, washed, attached onto poly-l-lysine-coated coverslips and examined at the indicated temperatures. Dotted line at 42 °C indicates RBC periphery. Domains are more visible at 20–30 °C. All scale bars 2 µm. B Morphometry of domain abundance. Number of theta*-labeled domains was determined per hemi-cell surface at the indicated temperatures and is expressed as percentage of 20 °C. Values are mean ± SEM of domains recorded on 125–634 cells (except at 10 °C, 9 cells) from two independent experiments. C Binding of theta* to RBCs. RBCs were labeled in suspension with theta* at the indicated temperatures. After centrifugation, pellets containing RBCs were analyzed by Western blotting for the His-tag on theta*. Arrowhead (right) corresponds to the expected position of intact theta*. D Western blot quantification of theta* binding using ImageJ software, expressed as percentage of 20 °C. For temperature dependence of domain size, see Fig. S6 and for “on-stage” effect of temperature, see Fig. S7