Modulation of GSK-3β Activity in Venezuelan Equine Encephalitis Virus Infection
Figure 4
BIO and BIOder inhibit GSK-3β in VEEV infected cells.
A) U87MG astrocytes and B) Vero cells were pretreated for 2 hours with DMSO, BIO (0.1 and 1.0 uM), or BIOder (0.1 and 1.0 uM), infected with VEEV TC-83 at MOI 0.1, and post-treated with DMSO, BIO, or BIOder. Mock infected cells were treated with DMSO, 1 uM BIO, or 1 uM BIOder. Twenty-four hours post infection cells were collected and protein extracts prepared. One mg of extract was IPed at 4°C overnight with GSK-3β antibody. The next day complexes were precipitated with A/G beads for two hours at 4°C. IPs were washed twice with TNE buffer and kinase buffer. Phosphorylation reactions were performed with IP material and 200 ng of glycogen synthase peptide 2 (Millipore) as substrate. Following incubation, samples were separated by SDS-PAGE, dried and subjected to analysis using Molecular Dynamics Phosphor Imager software. C) Immunoprecipitation of GSK-3β and IgG control from titration of extracts followed by staining. Constant amount of anti-GSK-3β or IgG Rabbit antibodies (10 µg each) were used for overnight precipitation using 10,100, and 1000 µg of VEEV infected U87MG extracts. Samples were IPed overnight and next day Protein A and G were added, washed in TNE150 and 0.1% NP-40 and then ran on a 4–20% followed by staining with coomassie blue. A dominant band of GSK-3β along with both heavy and light chains are stained.