Figure 2.

PEX12 interacts with PEX5. (A) Results of two-hybrid studies between PEX12 and PEX5. Two-hybrid reporter strains expressing the indicated fusion proteins were transferred to a nitrocellulose filter, submerged in liquid nitrogen to lyse the cells, and assayed for β-galactosidase activity. AD, GAL4 activation domain; and BD, GAL4-binding domain. (B) Filter binding experiments with PEX12 and PEX5. Equal amounts of MBP-LacZα and MBP-PEX12C were spotted on membranes and probed with [³⁵S]PEX5S (upper panel) or [³⁵S]PEX5L (lower panel). (C) PEX5 coimmunoprecipitates with PEX12/3xmyc. Lysates were prepared from fibroblasts that had been transfected with either pcDNA3-PEX12 or pcDNA3-PEX12/3xmyc. After immunoprecipitation with anti–myc antibodies, the immunoprecipitates were analyzed by immunoblot with anti-PEX5 antibodies (upper panel). In addition, equal amounts of the crude lysate before immunoprecipitation were assayed for PEX5 levels by immunoblot (lower panel). (D) PEX12 interacts with the PTS1-binding domain of PEX5. Two-hybrid reporter strains expressing the indicated fusion proteins were transferred to a nitrocellulose filter, submerged in liquid nitrogen to lyse the cells, and assayed for β-galactosidase activity.