Protein kinase C-mediated proteasomal degradation of MAP kinase phosphatase-1 contributes to glutamate-induced neuronal cell death

JCS02837 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. HT22 cells were exposed to 5 mM glutamate (Glu) or vehicle for 7 hours in the presence or absence of 20 mM SP600125 (SP). Phosphorylation of Jun (c-Jun) in whole cell extracts was determined by immunoblotting with an antibody against phospho-Jun (pc-Jun). Representative immunoblots are shown.

• Supplemental Figure 2 - Fig. S2. (A) ROS production after exposure to glutamate. 5 mM Glutamate was added to cells for the indicated times, and loaded with 5 mM DCF-DA to measure ROS. Each bar represents the amount of ROS accumulated during glutamate exposure for the indicated times. Net increases in ROS production are presented as closed bars. Three groups of cells were randomly selected from each sample, and the relative fluorescence intensity for each cell was measured and then averaged. Each group contained at lease 60 cells. **P<0.01, compared with zero time. (B) The effect of the ROS scavenger NAC on ROS generation, PKCd activity, and the level of MKP-1 and ERK1/2 phosphorylation (p) under glutamate exposure. (i) Cells treated with or without 5 mM glutamate (Glu) for 9 hours in the presence or absence of 20 mM NAC were loaded with 5 mM DCF-DA, and then analysed by confocal microscopy. (ii) Cell lysates were prepared, followed by PKCd immune complex kinase assay and western blot analysis. Representative images and immunoblots are presented.