Fig. 1.

ALDH1A1 is a disease-specific target of Cdk5. a ALDH1A1 is a direct substrate of Cdk5. Cdk5-p25 complex was subjected to kinase assay with either [³²P]ATP alone (lane 2) or with 6× His-ALDH1A1 and [³²P]ATP (lane 3) for 15 min. Lane 1 shows ALDH1A1 incubated with [³²P]ATP. b Cdk5 and ALDH1A1 associate under neurotoxic conditions in HT22 cells. Cdk5 was immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of Cdk5 and ALDH1A1 was analyzed (top panel). Lower panel shows Cdk5 input from total cell lysate. Each experiment was repeated at least three independent times. c Cdk5 and ALDH1A1 association under normal and neurotoxic conditions in HT22 cells. ALDH1A1 was immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of ALDH1A1 and Cdk5 analyzed. Lower panel shows ALDH1A1 levels from total cell lysate. d Cdk5 binds ALDH1A1 directly. ALDH1A1 and Cdk5 association was analyzed using recombinant Cdk5 and ALDH1A1 in an in vitro pull-down assay. 6× His-Cdk5 on beads was incubated with ALDH1A1 and their binding analyzed. e Cdk5 and ALDH1A1 bind directly. ALDH1A1 on beads was incubated with recombinant Cdk5 and their binding analyzed in an in vitro pull-down assay. f Glutamate stimulates the association of p35/p25 with ALDH1A1. p35/p25 were immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of p35/p25 with ALDH1A1 analyzed. Each experiment was repeated at least three independent times. g Glutamate stimulates association of p35/p25 and ALDH1A1. ALDH1A1 was immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of ALDH1A1 and p35/p25 was analyzed. Each experiment was repeated at least three independent times. h ALDH1A1 does not bind with p35 or p25 directly. ALDH1A1 and p35 or p25 association was analyzed using recombinant p35, p25, and ALDH1A1 in an in vitro pull-down assay. Recombinant ALDH1A1 on beads was incubated with 6× His-p35 or 6× His-p25 and binding analyzed. i Cdk5 associates with p35/p25 upon glutamate stimulation. p35/p25 were immunoprecipitated from either control or glutamate-treated HT22 cells (for 0–6 h), and the association of p35/p25 with Cdk5 analyzed. Each experiment was repeated at least three independent times. j Cdk5 associates with p35 and p25 upon glutamate stimulation. k Cdk5 activity increases upon glutamate treatment in HT22 cells. Cdk5 kinase assay was performed as described in the “Materials and Methods” section. Each experiment was repeated at least three independent times. *p < 0.05, compared with untreated HT22 cells