Figure 1.

Generation and validation of the Parkin^S65A/S65A mouse. (a) Depiction of targeting strategy to generate a constitutive knock-in of Ser65Ala (S65A) point mutation in the Mus musculus Park2 (Parkin) gene (targeting strategy based on NCBI transcript NM_016694.3). Exon 1 contains the translation initiation codon. The S65A mutation was introduced into exon 3. The Park2^S65A/S65A knock-in (KI) allele was generated following Flp-mediated recombination. (b) Rostro-caudal expression analysis of Parkin protein expression in the mouse central nervous system (CNS) under basal conditions. Immunoblot analysis of sub-dissected CNS regions from adult wild-type and Parkin^S65A/S65A mice. OB, olfactory bulb; CTX, neocortex; THAL, thalamus; STR, striatum; HC, hippocampus; VM, ventral midbrain; CB, cerebellum; BSt, brainstem; SPc, spinal cord. (c) Parkin Ser65 phosphorylation is essential for Parkin activation in primary neurons. Mature (21 DIV) primary cortical neuron cultures were established from wild-type and Parkin^S65A/S65A mice, and stimulated for 3 h with a combination of antimycin A (10 µM)/oligomycin (1 µM). Whole-cell extracts were subjected to SDS-PAGE and immunoblot analysis with anti-Parkin, anti-phospho-Ser65 Parkin, anti-CISD1 and anti-β-III tubulin antibodies.