Figure 3
H2B R95A mutant drastically reduces the expression of the STE5 gene and its encoded protein. (A) Relative mRNA expression of STE5 gene by qPCR analysis. Total RNA was isolated from the H2B WT and H2B R95A strains and 0.5 µg was used for reverse transcribed using the yeast RiboPure kit. The cDNAs were subjected to qPCR analysis with indicated primers (see “Materials and methods”). The ACT1 gene was used as a control. The data are representative of four biological replicates and analyzed by student t-test. ***Is equivalent to P-value < 0.001. (B) Immunoblot analysis of Ste5-GFP and GFP-Apn1 in the H2B WT and H2B R95A. Plasmids expressing either Ste5-GFP or GFP-Apn1 were introduced into the H2B WT and H2B R95A. Total cell extracts were prepared from these strains with the trichloroacetic acid method (see “Materials and methods”) and process for immunoblot analysis. The blot was probed with anti-GFP antibodies (upper panel). The lower panel was stained with Ponceau to monitor for equal protein loading. M, prestained protein markers in kDa. Arrows indicate the position of the GFP-tagged proteins, and the asterisk indicates a fragmented species of Ste5-GFP. (C) The expression level of the full-length Ste5-GFP in the H2B R95A mutant was quantified from panel (B) and expressed relative to the level detected in the H2B WT strain.
