Fig. 2.

Btn1p is regulated by the extracellular pH. (A) Expression of Btn1p measured in whole cell extracts from cells expressing Btn1p-V5. 50 μg of protein was run on SDS-PAGE and immunoblotted using an anti-V5 antibody (1:5000). (B) Btn1p levels in cells grown at pH 6.0 were approximately 1.7-fold higher than in those grown at pH 4.0 as determined by densitometry of Btn1p bands. (C) 50 μg of whole cell lysate from cells expressing Btn1p-V5 was harvested and treated for 2 hours with 1000 units PNGase F, followed by SDS-PAGE and immunoblotting. (D) Immunoblotting for Btn1p glycosylation mutants demonstrates that loss of Btn1p glycosylation occurs when N206, N175, N206 and all three putative asparagines are mutated. Actin was used as a loading control.