Heat shock treatment and protein synthesis inhibition effect on the stability of α-tubulin transcripts. For total transcription inhibition, promastigotes cultured at 26°C were incubated with 10 μg/ml sinefungin (Sinef) five minutes previously to the addition of 10 μg/ml actinomycin D (Act D). Afterwards, cultures were incubated either at 26°C (A) or 35°C with 5% CO2(C) for 0, 0.5, 1, 2, and 4 hours. For protein synthesis inhibition, 20 μg/ml of cycloheximide (CHX) was used in promastigotes cultured either at 26°C (B) or 35°C with 5% CO2(D) for 0, 0.5, 1, 2, and 4 hours. Finally, total RNA was extracted, and 8 μg from each sample were analyzed by Northern blotting. The blots were hybridized with α-tubulin ORF probe. Before transferring, gels were stained with ethidium bromide and photographed; rRNA staining was used as normalizing signal for densitometric measurement. For each series, densitrometric graphs (bottom panels) were standardized against the RNA signal found at time 0 (26°C), which was arbitrarily set as 1.0.
Ramírez et al. BMC Genomics 2013 14:454 doi:10.1186/1471-2164-14-454