Figure 2
Changes in the expression of p35/p25 in the cytoplasm and nucleus, and changes in the expression of CDK5R1 after transfection with miR-152. Effects of activation and inhibition of calpain on p35/p25, and subsequent changes in CDK5 activity in the cytoplasm and nuclear fractions were analyzed by immunoblotting (a) and protein expression quantification (b). The cytoplasmic fraction was normalized using GAPDH, and the nuclear fraction was normalized using Lamin B1 as reference. In the nuclear fraction, p25 was increased by calpain activation and p35 was absent, whereas nuclear p25 was decreased by calpain inhibitor. Cytoplasmic p25 was formed from the cleavage of p35 by calpain activation and inhibition. Effects of miR-152 mimic transfection and CDK5R1 knockdown on p35/p25 and CDK5 in the cytoplasm fractions were analyzed by western blot analysis (c) and band concentration quantification (d). Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies. The Input was normalized using GAPDH, and the proteins immunoprecipitated with CDK5 were normalized to CDK5. Western blotting analysis of p35/p25 levels in SKES-1 cells transfected with miR-152 and CDK5R1 siRNA in nucleus fraction (e). Immunoprecipitation (IP) was performed with CDK-5, followed by western blotting with the indicated antibodies. Quantification (f) of changes in the protein expression of co-precipitated p35/p25 in the nuclear fractions were performed. The Input of nucleus fraction was normalized using Lamin B1, and the proteins immunoprecipitated with CDK5 were normalized to CDK5.
