FIG. 5.

SDS-PAGE Western blot of laccase of cellular fractions of C. neoformans. Laccase-containing cryptococcal cell suspension from either H99 or B-3501 cells were either boiled in 1% SDS (lane 1) or centrifuged, and the supernatant (lane 2) and the pellet (lane 3) were separately subjected to electrophoresis without boiling. Alternatively, centrifuged fractured cell pellets were extracted sequentially with 1 M NaCl, 6 M urea, and 1% SDS and either subjected to electrophoresis without boiling (lane 4) or with boiling in 1% SDS (lane 5). Salt-urea-SDS-extracted fractured cell pellets were additionally treated with 30 mM NaOH at 37°C for 30 min (lane 6) or 100 mM DTT (lane 7) or 2% β-mercaptoethanol (lane 8) and then electrophoresed without boiling. Each lane contained approximately 20 μg of carbohydrate; in addition, lanes 1 and 2 contained 10 μg of protein.