Glutathione bead

Purified GST-EhAK1 was allowed to bind Glutathione beads (Amersham) for 1 h at 4°C in 1% PBS/BSA/0.1% tween-20. EhCaBP1 or EhCaBP1ΔEF was then added to the reaction and the reaction was incubated for 2 h at 4°C. The beads were then washed thrice with 1% PBS/BSA/0.1% tween-20, and twice with PBS. Bound proteins, eluted by adding 2× SDS–PAGE buffer, were analysed by western blotting. The same procedure was followed for other proteins with GST-tags.

Chimeric EC2 fusion proteins were produced by overlap extension PCR, with the swapped regions described in S1 Table in S1 File. All of the wild-type and point mutant EC2 fusion proteins, except Y148M and Y148A, were previously described [25] (link); the Y148 mutants were produced in the same way. The production and characterisation of the tetraspanin EC2 GST fusion proteins has been described in detail previously [25] (link). Briefly, tetraspanin EC2 regions that had been cloned into the pGEX-KG vector were expressed in Rosetta Gami B(DE3) E. coli (Merck Biosciences), culturing at 37°C for 4 hours after IPTG induction. Cells were pelleted and lysed by sonication in the presence of a protease inhibitor cocktail. Recombinant protein was purified in a single step by affinity chromatography on glutathione beads (Amersham-Pharmacia). Protein purity was analysed by Coomassie staining of SDS-PAGE gels and total protein concentration determined by Bradford assay. As it was not possible to separate the full-length EC2 fusion protein from the smaller fragments produced, the percentage of full length material in each sample was measured by densitometry. This was plotted against the inhibitory activity of each sample to ensure that inhibition of MGC formation was not a simple function of the concentration of the full length fusion protein (S1 Fig. in S1 File).

In vitro acetyltransferase assays were performed as previously described and detected by using western blotting with anti-acetyl lysine antibody [26 (link)]. Briefly, recombinant GST-Lin28B and GST-PCAF-HAT2 proteins were expressed in E. coli BL21 and purified using glutathione beads (Amersham Biosciences). For in vitro acetyltransferase assays, 1 μg of GST-PCAF-HAT2 and 5 μg of GST or GST-Lin28B were incubated in 25 μl of acetyltransferase assay buffer (50 mM Tris, pH 8, 10% glycerol, 10 mM butyric acid, 0.1 mM EDTA, 1 mM DTT, and 1 mM PMSF) with 20 μM acetyl CoA at 30 °C for 1 h. The reaction products were separated via 15% SDS-PAGE and analyzed by western blotting with anti-acetyl lysine antibody.

Purified GST-EhARPC1 was incubated with Glutathione beads (Amersham) for 1 h at 4°C in buffer comprising of 10mM Tris-Cl (pH 7.5), 0.1mM EDTA, 0.1% NP-40 (w/v), 2mM DTT, 100mM NaCl, 0.2mM PMSF. Then EhCaBP1 or his tagged proteins as indicated was added to the reaction and incubated for 2 h at 4°C. The beads were then washed with washing buffer comprising of 10mM Tris-Cl, 1% Glycerol, 1mM EDTA, 0.1% NP-40, 2mM DTT, 100mM NaCl, and 0.2mM PMSF thrice. Protein was eluted by adding 2X SDS–PAGE buffer followed by boiling for 5 min. The proteins were then analysed by western blotting. The same procedure was followed for other proteins with GST-tags.