Properties of human claudins produced by the S-MF method.
(a) SEC elution profiles of human claudins, prepared by the S-MF method. The peak fractions of SEC were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. (b) Comparison of the properties of claudin-4 produced by the P-MF and S-MF methods (lanes 1–8 and 9–14, respectively). The fractions in the purification steps were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. (c) SPR binding analysis of the cell-free produced claudin-4 against the immobilized GST-tagged C-CPE on the sensor chip. (d) Conceptual diagram of the thermal stability and folding analysis of a membrane protein by the CPM assay. (e) Thermal stability analysis of claudin-4 (open symbols) and the claudin-4∙C-CPE complex (closed symbols) by the CPM assay, in the absence (circles) or presence (triangles) of 1 M guanidine-HCl (GnHCl) at 40 °C. (f) TLC analysis for the detection of the lipids bound to claudin-4 in the purified claudin-4∙C-CPE complex sample. (g) The CPM assay for the assessment of the thermal stabilization effect of PC for the claudin-4∙C-CPE complex. (h) The CPM assay for the assessment of the thermal stabilization effect of CHS for the claudin-4∙C-CPE complex. The t1/2 values in the absence (open circles) and presence (closed triangles) of 1 M GnHCl at each concentration were plotted in this graph. The gel images and graphs are representatives of at least two experiments ((a–c), (e,f)) or one experiment (g,h). The gel images of (a) were processed by cropping.