Fig. 3. Two-dimensional western blot of -tubulin from S100 extracts and western blot analysis of -tubulin produced from transcription/translation reactions. (A) Jurkat S100 (30 µg) was incubated with or without purified grB (50 µM), as indicated. The reactions were then separated on a first dimension IEF IPGphor strip with pH range of 4-6. The second dimension was 12% SDS-PAGE and the presence of -tubulin was determined by western blotting with anti--tubulin. (B) -tubulin containing FLAG-tagged wild-type amino acid sequence (WT) or point mutants D424A, D431A or D438A, as indicated were created by TNT reactions of the corresponding plasmids. Reactions were incubated with (+) or without (–) purified grB (50 µM). Protein products were separated on 12% SDS-PAGE for western blot analysis with anti-FLAG.