Figure 10. GCN4 transcripts and Gcn4 protein relative levels are significantly increased rpc128-1007 yeast cells and moderately in maf1Δ cells.

Yeast cells were grown in rich medium supplemented with 2% glucose until reached exponential growth phase (D₆₀₀ ≈ 1.0). SYBR-Green-based real-time PCR (A) showed that GCN4 transcript increased 2-fold in maf1Δ and by 11-fold in rpc128-1007. Wild-type strain expression level was taken as 1.0. Samples were normalized to two reference genes — U2 spliceosomal RNA (U2) and small cytosolic RNA (SCR1). Asterisk (*) indicates P-values lowered than 0.05 according to Student's t-test using all technical replicates of biological samples. Western blotting assay (B) showed increased stability of Gcn4-3HA protein in maf1Δ and rpc128-1007 mutant strains expressing chromosomally encoded Gcn4-3HA. Total cell protein extracts were subjected to SDS–PAGE and examined by Western blotting with anti-HA antibodies (B). Quantitative relative level of Gcn4-3HA protein in comparison with yeast Vma2 protein level was calculated for at least three independent biological replicates (C).