Figure 3.

Parkin antibodies epitope mapping studies. A. Schematic of parkin protein. The numbers indicate the amino acid residues that correspond to the respective protein domains. Ubl is the ubiquitin-like domain, Mid is the linker domain, R1 is the first RING finger domain, IBR is the in-between-ring-finger domain, and R2 is the second RING finger domain. B. Full-length human parkin cDNA corresponding to amino acids 1–465 and various truncated cDNA fragments of parkin were cloned in the bacterial expression vector pDEST 15, that expresses N-terminal GST-tagged recombinant proteins. The recombinant proteins were expressed in BL21 E. coli cells. The cells were harvested and equal amounts of total protein lysates were analyzed by western blot with a GST antibody in order to confirm similar expression of the respective proteins. Additionally, equal amounts of the protein extracts were assessed for immunoreactivity with the PRK8, PRK28, and PRK109 antibodies. The experiments were performed in duplicate. Representative blots are shown for each construct. The identical blot for PRK28 is shown at different exposure times in order to reveal the faint immunoreactivity of the 1–399 construct that is not readily detectable at the shorter exposure time. The mobility of molecular mass markers is indicated on the left.