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. 2013 Mar 6;8(3):e58956. doi: 10.1371/journal.pone.0058956

Figure 3. Construction of the constitutive KAR2 expression strain in C. neoformans.

Figure 3

(A) The strategy for the construction of the PH3 :KAR2 strain containing the NEO resistance marker (NEO R) and the histone H3 gene promoter (H3 pro). (B) Northern blot analysis for measuring KAR2 expression in PH3 :KAR2 strains [YSB1751 (lane 2), YSB1752 (lane 3), YSB1741 (lane 5), YSB1744 (lane 6), YSB1745 (lane 8), and YSB1746 (lane 9)] and their parent strains [WT H99 strain (lane 1) and ire1Δ (lane 4) and hxl1Δ (lane 7) mutants]. KAR2 expression levels were quantitatively measured with a phosphorimager and normalized to ACT1 expression levels. Each KAR2/ACT1 is a value relative to that of the WT strain set to 1.0. (C) Northern blot analysis for measuring KAR2 expression in WT, ire1Δ, hxl1Δ mutants, and PH3 :KAR2 strains (YSB1751, YSB1741, and YSB1745) treated with or without TM (0.3 μg/ml) for 1 h. (D) RT-PCR analysis of UPR-induced HXL1 splicing with cDNA samples in WT H99 strain and PH3 :KAR2 strain (YSB1751) treated with or without TM (0.3 μg/ml) or DTT (20 mM). RT-PCR of HXL1 and ACT1 was performed with gene-specific primers listed in the Materials and Methods.