Activation of Cyclin-Dependent Kinase 5 Is a Consequence of Cell Death
Figure 1
Cdk5 activation is independent of Bim and cathepsins. (a) Pregnant female mice were injected intraperitoneally as described in Section 2. Cdk5 activation by immunohistochemistry showed many cells with Cdk5 activation labeled by Cdk5 antibody in bim and bim mouse embryos with or without CP treatment, 400X. (A) Positive signals were counted in 3–5 fields of 400, and the number of positive signals per field was used for plot and analyzed by t-test. (B) The error bars represent the standard deviation from at least three individual experiments ( ). (b) Wild type and cathepsin B, D, and L cells were incubated with CPT (50 M). (A) At 24 and 72 hours, the amount of cell death was determined by trypan blue exclusion. The error bars represent standard deviation obtained from at least 3 independent experiments. Protein samples were isolated from cells with RIPA buffer. (B) Western blot analysis of equal amounts of cell lysates from cells using Cdk5 antibody showed an unchanged level of Cdk5 protein (32 kDa) during cell death. (C): Equal amounts of cell lysates from wild type and cathepsin B, D, and L cells incubated with CPT (50 M) for 72 hours were used for western blot using p35 antibody. These cells demonstrated an induction of p25 during CPT-induced cell death in the absence of the cathepsins. (D) Histone H1 kinase activity induced during cell death was detected in Cdk5 immunoprecipitates in wild type cells: cathepsin B cells, cathepsin D cells, and cathepsin L cells following 72 hours treatment with CPT. The histone H1 phosphorylated by Cdk5, as detected by western blot, increased corresponding to cell death in controls and knockouts. Cdk5 is a relatively stable protein in cells and the expression of Cdk5 by western can be used as a loading control [20, 25]. Scale mark equals 100 M.
