FIGURE 1.

Purification of MAT2β V1 and V2 and identification of interacting proteins. A, V1 and V2 were overexpressed in Rosetta pLysS cells and purified by nickel-nitrilotriacetic acid. The SDS-PAGE gel illustrates His-tagged V1 and V2 proteins expressed with isopropyl 1-thio-β-d-galactopyranoside induction at 37 °C or 16 °C. 5 μl of V1 or V2 beads was heated in SDS loading buffer and run on a 12% SDS-PAGE gel. Proteins purified by nickel-nitrilotriacetic acid were visualized with Coomassie Blue staining. B, Western blot analysis for His tag was performed to confirm His-tagged V1 protein expressed at 37 °C and V2 protein expressed at 16 °C. C, SDS-PAGE gel demonstrating proteins binding with V1 or V2. Pulldown experiments were performed using RKO cell lysate and His-tagged V1 or V2. 20 μg of binding proteins and 5 μl of V1 or V2 beads were separated by 12% SDS-PAGE and visualized with silver staining. D, Western blot analyses of MAT2A and MAT2β-encoded proteins, which were present in the eluted proteins after V1 or V2 pulldown assay but not in the control (His-tagged c-Myc expression vector).