Figure 6.

NHL3-HA is highly enriched in the upper phase of two-phase partitioning fraction. Two-phase partitioning was used to separate plasma membrane and internal membranes. Five micrograms of proteins from microsomal (M), upper phase (U), and lower phase (L) preparations was loaded with 2 μL of total (T) extracts (approximately 30 mg of ground tissue boiled in 200 μL of SDS buffer) and was subjected to western blotting. Antibodies specific for amino acids 6 through 51 of AHA2, a plasma membrane-localized proton ATPase, and against the plant-specific endoplasmic reticulum lumenal binding protein BiP were used to show the efficiency of the fractionation process. Identical results were obtained in two different transgenic lines, and only a representative experiment is shown.