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. Author manuscript; available in PMC: 2013 Jun 10.
Published in final edited form as: Virology. 2011 Apr 22;415(1):56–68. doi: 10.1016/j.virol.2011.03.025

Figure 5. 6BIOder does not inhibit cellular gene expression in the absence of Tat and is specific to GSK-3β.

Figure 5

A) Total RNA was isolated from cells treated with 6BIOder (1μM) using Trizol. RNA was treated with 0.25 mg/ml DNase I for 1 hour, followed by heat inactivation at 65°C for 15 minutes. A total of 1 μg of total RNA was used to generate cDNA with the iScript cDNA Synthesis kit using oligo-dT reverse primers. Primers for PCR were MCL-1, IL-8, cyclin D1 and GAPDH as control. B) Two mg of U937 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 run on a 4-20% SDS-PAGE, dried and subjected to analyzed using Molecular Dynamics Phosphor Imager software.