Anti myc antibody

GST or GST-tagged ACBD3 constructs were cotransfected with Dexras1-Myc constructs into HEK293T cells using PolyFect (Qiagen, Valencia, CA), with a transfection efficiency of greater than 90%. Cells were lysed 48 hr after transfection in buffer A (100 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 15% glycerol, 1 mM PMSF, 25 mg/ml antipain, 50 mg/ml leupeptin, 50 mg/ml aprotinin, 25 mg/ml chymostatin, and 25 mg/ml pepstatin). Lysates were precleared with pansorbin cells (Calbiochem, Billerica, MA), then 1 mg of total protein was incubated with Glutathione-Sepharose beads overnight at 4 C. Beads were washed with wash buffer (50 mM Tris [pH 7.4], 500 mM NaCl, 10 mM b-glycerophosphate) twice, then once with buffer A. Beads were quenched in sample buffer (100 mM Tris [pH 6.8], 10% glycerol, 250 mM b-mercaptoethanol, 2% sodium dodecyl sulfate, and bromophenol blue). Total protein (50 mg) was loaded as input. Rhes-Myc binding was examined using an anti-myc antibody (EMD Millipore, Billerica, MA) followed by incubation with anti-mouse secondary conjugated to horseradish peroxidase (HRP) (Jackson Immunoresearch Laboratories, West Grove, PA); blots were then stripped and probed with an anti-GST antibody conjugated to HRP to detect ACBD3. Pierce Chemiluminescence (Life Technologies, Grand Island, NY) was used to detect bands on the Western blot.