Ras1 Acts through Duplicated Cdc42 and Rac Proteins to Regulate Morphogenesis and Pathogenesis in the Human Fungal Pathogen Cryptococcus neoformans
Figure 1
Ras1 is required for Cdc42/Rac-mediated PAK activation.
(A) The ras1Δ mutant strain and a wild type strain (H99) were transformed with plasmids constitutively expressing GFP-CDC42 and GFP-RAC2. Representative transformants were grown overnight, diluted into fresh media, and incubated at 30°C or 37°C for four hours. Cell lysates were prepared, quantified, and divided between a loading control sample and an experimental sample. Each experimental sample lysate was incubated with GST-PAK-PBD to specifically bind GTP-bound, active Cdc42 or Rac2 and precipitated using glutathione beads. Experimental (top panel) and control (bottom panel) samples were separated by NuPAGE electrophoresis and transferred to PVDF. Immunoblot analysis with anti-GFP antibody revealed bands that migrated at the predicted molecular weight for GFP-Cdc42 and GFP-Rac2 (50 kDa, indicated in each panel). Doublets in the experimental samples represent known changes in Cdc42 mobility following PAK analysis. (B) Signal intensity ratios were determined using ImageJ.