FIG. 8.

Formation of a holo-Shp/holo-HtsA complex. (A) Native PAGE analysis of a holo-HtsA/holo-Shp mixture. Holo-HtsA (60 μM) was incubated with holo-Shp at the indicated holo-HtsA:holo-Shp molar ratios in 20 mM Tris-HCl, pH 8.0, at room temperature for 30 min, mixed with an equal volume of 2× native sample buffer, and resolved by PAGE under native conditions. A mixture of 120 μM holo-Shp and 60 μM SPy0252 was also analyzed as a control. Holo-HtsA (rHtsA), holo-Shp (hShp), and SPy0252 (252) were included as references. The numbered boxes indicate the pieces of gels from which proteins were extracted for SDS-PAGE analysis shown in panel B. (B) SDS-PAGE analysis of selected bands from panel A. Pieces of gel indicated by the numbered boxes in panel A were excised. Proteins in these gel pieces were extracted by boiling in 20 μl of 1× Laemmli sample buffer for 4 min and resolved by SDS-PAGE through a 12% polyacrylamide gel. Lane numbers correspond to the numbers of the boxes in panel A for the bands where proteins were extracted. Both gels were stained using GelCode blue.