Figure 6.

Analysis of calcineurin in Kloch1-1 cells. (A) Northern Blotting analysis of KlCNB1 WT and Kloch1-1 cells. The same amount of total RNA (40 μg) from the strains was loaded on each lane; the ethidium bromide-stained gel of the autoradiogram is shown in the bottom part of the panel and the mRNA loading was normalized using the 26S rRNA bands. Quantification, by densitometric analysis, of the radiolabeled signal on the blot is shown in the right part of the panel. The hybridization signal for wild type strain was set as 1. (B) RT-PCR semi-quantitative analysis of KlCNA1 gene. Exponentially growing wild-type and Kloch1-1 cells were collected and RNA was isolated. Reverse transcription and PCR reactions with the specific primers described in materials and methods was performed. Shown is the electrophoresis in a 2% agarose gel of 10 μl of each PCR reaction with 5 μl (1×) and 10 μl (2×) of cDNA as template. RT-PCR of the KlUBC6 gene was performed as an internal control. RNA extraction was performed twice and the results shown are representative of four independent RT-PCR experiments. (C) Growth of indicated yeast strains onto solid medium supplemented with 20 mM EGTA.