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Fig. S1 Raf constructs exhibit appropriate kinase activity. (a) Active B-Rafs (myc-B-Raf V600E, full-length form and myc-B-Raf CAT, truncated form), inactive B-Raf (myc-B-Raf KD, full-length form), and GFP transfected to 293T cells. Exogenous B-Rafs were immunoprecipitated using myc antibody. Raf kinase assay was performed with GST-MEK. Western blotting analysis of the reaction product was performed using a phospho-MEK antibody. The same blot was reprobed with myc to show exogenous protein pull-down and with MEK antibodies to show equal substrate amount in each lane. Western blot of an aliquot of pre-immunoprecipitated cell lysates with a tubulin antibody serves to show equal amount of input lysate used. (b) Active c-Raf (HA-&Dgr; c-Raf, truncated form), inactive c-Raf (HA-&Dgr; c-Raf KD, truncated form and HA-Raf KD, full-length form), and GFP transfected to 293T cells. Exogenous c-Rafs were immunoprecipitated using HA antibody. Raf kinase assay was performed with GST-MEK. Western blotting analysis of the reaction product was performed using a phospho-MEK antibody. The same blot was reprobed with HA to show exogenous protein pull-down and with MEK antibodies to show equal substrate amount in each lane. Western blot of an aliquot of pre-immunoprecipitated cell lysates with a tubulin antibody serves to show equal amount of input lysate used.

Fig. S2 ATF-3 induced neuronal apoptosis is confirmed by TUNEL staining. Cerebellar granule neurons were transfected with an expression plasmid encoding Flag-tagged ATF-3 and then switched to HK or LK medium. The cultures were processed for TUNEL-staining (to identify apoptotic neurons), ATF-3 immunocytochemistry (to identify transfected neurons), and DAPI-staining (to visualize nuclear morphology) 24 h after transfection. The proportion of ATF-3-positive neurons (TXRed) that were also TUNEL-positive (green) was quantified. There was almost complete overlap between TUNEL-positive cells and cells with apoptotic nuclei as judged by DAPI-staining.

Fig. S3 3-NP induces striatal degeneration. Cresyl violet staining of brain sections from 50 μm coronal sections from control and 3-NP treated mice. 3-NP injected animal display selective striatal degeneration as evidenced by reduced cresyl violet staining. Doses and conditions of administration are detailed in Experimental procedures.

Fig. S4 MEK inhibitors do not block neuroprotective compound-mediated neuronal survival. (a) Cerebellar granule neurons were treated for 3 h with HK, LK, or LK medium containing the neuroprotective drugs. Lysates from the cultures were subjected to western blotting using phospho-MEK and phospho-ERK antibodies. The same blot was reprobed with a tubulin antibody to demonstrate comparable loading. (b) Cerebellar granule neurons were treated with HK, LK, and LK medium plus the various neuroprotective compounds in the absence or presence of MEK inhibitors, 40 μM PD 89059 (PD) or 20 μM U0126 (U0). Neuronal viability was assayed 24 h later using DAPI staining. Data indicates the viability of neurons with mean survival and standard deviation. Survival was normalized to cultures treated with HK.

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FilenameFormatSizeDescription
JNC_5226_sm_Supplementary Figure 1.tif770KSupporting info item
JNC_5226_sm_Supplementary Figure 2.tif617KSupporting info item
JNC_5226_sm_Supplementary Figure 3.tif4446KSupporting info item
JNC_5226_sm_Supplementary Figure 4.tif637KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.