Fig. 3. Sla1p and Sla2p interact in vitro. (A) GST-Sla1p was purified as described in Materials and Methods. Cell extracts were passed over the protein on a column and proteins in bound and unbound fractions were separated by SDS-PAGE and transferred to PVDF for analysis. Western blots were probed with antibodies raised against Sla2p. Sla2p bound only to Sla1p carrying beads and not to control beads alone. FT, flow through; W1,W3, washes 1 and 3; B, bound. (B) To investigate whether the interaction between Sla1p and Sla2p was direct, His-tagged Sla2p was purified from cells as described in Materials and Methods, and its binding to GST control beads or to GST-Sla1p beads was assessed. As shown in the upper panel Sla2p only binds to Sla1p-containing beads. As a control, the extracts from KAY419 cells from which GST-Sla1p was purified were probed to demonstrate the presence of Sla2p in the extract. Then, following GST-Sla1p purification the beads were probed to demonstrate that no Sla2p had co-purified with the Sla1p. Therefore, the Sla1p-Sla2p binding observed was only caused by the presence of the added Sla2p to the GST-Sla1p beads. FT, flow through; W1,W3, washes 1 and 3; B, bound. (C) Bacterially expressed GST or GST-Sla1(118-511SH3#3) was incubated with His-tagged Sla2p as described in Materials and Methods. The beads were washed and the input material (I), wash (W) and bound (B) fractions were run on a gel that was then stained using Coomassie dye. The three bands marked x are all detected by western blotting with GST antibodies indicating that they are all Sla1 degradation products; bands marked were detectable using Sla2 antibodies.

Return to article