results were obtained in other experiments performed by scraping cells from plates in the absence of trypsin. UV Irradiation. Jurkat cells were plated in 100 15 mm 106 polystyrene petri dishes at a concentration of 2 cells/ml and 
irradiated in a Stratalinker 2400 at a distance of 9 cm for 30 s. After irradiation, cells were incubated at 37 C for the indicated times before harvesting. His-tagged ASF/SF2 was expressed in Escherichia coli Origami pLacI with IPTG induction, extracted using a Ni-NTA Spin kit, analyzed by BCA protein assay, Coomassie staining, and Western blotting using antibodies specific for both His-C tag, and HSV tag, respectively. In Vitro Transcription/Translation. methionine-labeled p35, IL-1, Ich-1, mouse SRPKs, human SRPKs, or Clk/Sty kinases 14 were in vitro transcribed and translated using the TNT rabbit reticulocyte lysate kit, according to the manufacturer’s instructions. Reactions were performed using 0.25 g plasmid in a 10 l transcription/translation reaction mixture containing 0.5 l of translation grade methionine. In Vitro Caspase Cleavage Assays. In vitrotranslated proteins synthesized as described previously were incubated in caspase cleavage buffer with recombinant caspases for 90 min at 30 C as described previously. cDNAs encoding individual caspases were a gift of H. Li and J. Yuan. The data for caspase-8 cleavage of SRPK1 was confirmed using recombinant, purified His-tagged caspase-8. Recombinant caspases were prepared as described and frozen at 80 C until used. In a separate reaction, the mixture was then GS1101 biological activity separated by SDS-PAGE, transferred to nitrocellulose and exposed for autoradiography. In separate experiments, each caspase was incubated with in vitrotranslated proteins including p35, procaspase 2, or IL-1 to confirm their activity. 1214 Identification of an Autoantigen Kinase Signaling Pathway Immunoprecipitation and Western Blot Analysis. Lysates were precleared once with 100 l of a 50% solution of protein A-Sepharose in detergent lysis buffer and 5 g rabbit antimouse IgG for 12 h. Mouse mAbs were used as follows: anti-SRPK1 and anti-SRPK2; anti-cdc2; anti-SC35; and anti-U2B . Human autoimmune serum samples were employed as follows: 3 l human polyclonal antiScl-70 or antiU1-snRNP. U1-snRNP-specific sera that were previously shown to coprecipitate SR proteins PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19803812 and control sera have been described previously. 2 l antiDNA-dependent protein kinase was used for IP kinase and Western blotting experiments. Immunoprecipitations were performed after addition of detergent lysis buffer to a total volume of 500 l, and rotation in a 4 C cold room for 24 h. Precipitates were harvested by centrifuging for 20 s at 12,000 rpm in a refrigerated Heraeus microfuge, washing three times with detergent lysis buffer, resuspending in SDS loading buffer with 9% 2-mercaptoethanol, boiling for 5 min, and separating by SDS-PAGE as described previously. Proteins were transferred to nitrocellulose for Western blotting experiments. Antibodies and dilutions used were as follows: anti-cdc2 ; antiDNA-PKCS; anti-SRPKs; anti-topoisomerase I is a synthetic compound that promotes the activity of pyruvate dehydrogenase by inhibiting its repressor protein called pyruvate dehydrogenase kinase. The activation of PDH leads to a reduction in ambient cellular lactate concentrations both in vitro and in vivo which contributes to the therapeutic use of DCA in the treatment of systemic lactic acidosis in humans. The therapeutic potential of DCA
