FIGURE 2.

In vitro interactions between OASTL and the SULTR1;2 STAS domain. A and B, in vitro binding assays show interaction of OASTL fused at its C terminus to His₆ (OASTL-His₆) with SULTR1;2 or SULTR1;1 STAS domain (with or without L region), which is fused to MBP at its N terminus (MBP-1;2STAS, MBP-1;2LSTAS, MBP-1;1STAS, and MBP-1;1LSTAS). An equal amount of OASTL-His₆ was co-incubated with MBP or the MBP fusion proteins at 22 °C for 1–4 h with gentle rocking; MBP or MBP fusion proteins were trapped on an amylose resin, which was washed three times with the reaction buffer (minus the proteins) prior to releasing the amylose bead-bound fractions (PPT, precipitate) and resolving the released proteins by SDS-PAGE followed by immunological detection using His₆- and MBP-specific antibodies. C, interaction of SULTR1;2 with OASTL. C-terminally 4MYC-tagged SULTR1;2 (SULTR1;2–4MYC) was transiently expressed in tobacco epidermal cells, and the microsomal fraction was co-incubated with N-terminally His₆-tagged OASTL (His₆-OASTL). An Ni-NTA resin was used to bind His₆-OASTL, and the unbound (U), the flow-through of the last wash (W), and the resin-bound (B) fractions were assayed for His₆-OASTL and SULTR1;2–4MYC by SDS-PAGE followed by immunological detection using His₆- and MYC-specific antibodies.