Western blot analysis and intracellular localisation of AtStp2 in S. cerevisiae. (A) Crude extracts of L-arabinose-grown TSY01 cells expressing either ScGal2 (lane 1), or HA-tagged AtStp2 (lane 2) were prepared, and 20 μg of total protein were separated by SDS PAGE. Western blotting was performed as described in Methods. The arrow indicates the HA-tagged AtStp2 protein. (B) For subcellular localization of AtStp2 crude extract of TSY01 cells grown on L-arabinose expressing HA-tagged AtStp2 was transferred on a seven step sucrose density gradient. After centrifugation the gradient was fractionated and the localization of AtStp2-HA in S. cerevisiae was analyzed via Western blot analysis. Antibodies against Pma1 (plasma membrane) and Dpm1 (endoplasmic reticulum) served as controls. HA: hemagglutinin.