Baculoviruses were made in High Five or SF9 insect cells from pFastBac plasmids using the Bac-to-Bac expression system (Invitrogen). Batch purification using glutathione resin beads (Novagen) was used to purify the GST-tagged proteins from infected High Five or SF9 insect cell cultures. Briefly, cells were rinsed with phosphate-buffered saline (PBS) and lysed in 50 mM Hepes (pH 7.5), 100 mM NaCl, and 1 mM dithiothreitol (DTT) (buffer A) with 0.1% Triton X-100, 100 μM phenylmethylsulfonyl fluoride (PMSF), 2 mM benzamidine, and leupeptin (50 μg/ml). The soluble lysate was incubated with glutathione resin beads for 30 min at 4°C. Protein-bound beads were washed three times in buffer A and then eluted three times in buffer A with 10 mM glutathione. Eluent was loaded in a 30-kDa Amicon Ultra centrifugal filter unit (Millipore) and washed or concentrated three times with buffer B [20 mM Hepes (pH 7.5) and 1 mM DTT]. Glycerol was added to 50% volume before measurement of GST-PKCζ concentration using bovine serum albumin standards on a Coomassie brilliant blue stained gel, and enzyme stocks were stored at −20°C.

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