Presence of novel GSK-3β complexes in VEEV infected cells.

<p><b>A</b>) Samples from both infected and uninfected cells were loaded on a sizing column and separated in presence of a 500 mM salt buffer. No detergents were used during the chromatography steps. Each fraction (over a range of 60 fractions) contained 500 µL and only 250 µL samples were precipitated, resuspend in low volume of TNE₅₀ and 0.1% NP-40 (10 µL) and ran on a gel for western blotting. Samples were western blotted for presence of GSK-3β and actin. <b>B</b>) GSK-3β IPs from fractions 31, 41, 51 (100 µL each) were mixed with GSK-3β Ab (5 µg) overnight and washed next day first with RIPA buffer (1×) and then TNE₅₀ and 0.1% NP-40 (2×) followed by kinase buffer (3×) prior to addition of substrate and ³²P-ATP. Samples were ran on a gel, stained, destained (over 4 hrs), dried and then exposed to phosphoImager cassette. Control immunoprecipitation from these fraction (50 uL each) were mixed and used for IP/Kinase using IgG (5 µg). <b>C</b>) IP/kinase reactions were treated with either BIO or BIOder (0.1 uM or 1 µM) <i>in vitro</i> during the kinase reaction. Samples were ran on a gel and exposed to phosphoImager cassette.