Figure 2.

Expression of CnMPR1-HIS tagged cDNA in S. cerevisiae. (A) Reverse transcriptase-PCR (RT-PCR) detected mRNA transcript levels of CnMPR1 in Sc. An Sc strain was transformed with a plasmid containing a C-terminal HIS-tagged MPR1 cDNA isolated from C. neoformans (Sc<CnMPR1^HIS>). Transcripts of MPR1 were not detected in a wild type strain of Sc (ScWT). The plasmid containing CnMPR1-cDNA, used as a positive control for PCR, indicated a cDNA size of ~2.1 kb similar to that detected for Sc<CnMPR1^HIS>. β-actin was used as an internal control for RT-PCR. (B,C) Western blot analysis of Mpr1^HIS in whole cell lysates of ScWT and Sc<CnMPR1^HIS> revealed expression of Mpr1 protein in Sc<CnMPR1^HIS>. (D–G) Indirect immunofluorescence further confirmed the expression of Mpr1 in Sc<CnMPR1^HIS>. Bright field images (D,F) and corresponding fluorescence images (E,G) are shown. (E) Mpr1^His was detected with a 6X-HIS primary antibody followed by a secondary antibody (Alexa Fluor 488) in Sc<CnMPR1^HIS>. (G) Mpr1 protein was not detected in ScWT.