Fig. 2.

Taxol analog 2-m-AzTax differentially photoaffinity labels distinct tubulin isotypes. Five micromolar BBT was incubated with 6 µM [³H]2-m-AzTax, in the presence of 1 mM GTP, at 37 °C for 30 min, followed by UV irradiation and centrifugation as described in Materials and Methods. (A) Separation of BBT by high-resolution IEF. Tubulin isotypes were identified by mass spectrometry. (B) Photolabeling of total α-tubulin and β-tubulin. (C) β-Tubulin isotypes were identified by CNBr digestion and mass spectrometry. Level of each isotype was determined by ImageJ and the amount of radioactivity associated with each band was determined by liquid scintillation counting as described in Materials and Methods. (D and E) Amount of radioactivity associated with βII- and βIII-tubulin (red bolded values in C) was normalized to their protein levels, and relative photolabeling of βII- and βIII-tubulin was plotted.