Figure 3.

Immunodetection of WT and S460C protein. (A) Western blot obtained from a pool of five oocytes injected either with water, WT, or S460C cRNA. 10 μl of the lysates was separated on a 9% SDS gel and, after blotting, immunoreactive proteins were visualized by incubation with an antibody against the rat NaPi IIa NH₂ terminus. This blot confirms that lysate from oocytes expressing S460C presents a similar band (97 kD) to the WT. (B) Streptavidin precipitation of oocyte lysate obtained from pools of five oocytes injected either with water, wild type, or S460C cRNA and incubated with 100 μM MTSEA-Biotin. Cells were then homogenized in 100 μl buffer (see materials and methods) and 90 μl of each lysate was incubated with Streptavidin beads. After washing, bound proteins were eluted with loading buffer (see materials and methods) and the elute was then treated as for A. Single band at 97 kD confirms that the alkylated protein was S460C.