FIG. 2.
TFE is selectively depleted under heat shock conditions, but transcription is not affected. (A) Sulfolobus cells growing at 76°C (lane1) were incubated at 0°C, 76°C, 80°C, 85°C, and 90°C (lanes 2 to 6, respectively) for 1 h. The cells were harvested and suspended directly in SDS-PAGE loading buffer. Western blots were probed with the indicated antibodies. (B) qPCR analysis of TFE, TFB1, and TBP mRNAs. Total RNA was isolated from 10 ml of cells incubated at 90°C for 0, 5, 10, 30, and 60 min and converted into cDNA. PCR amplification of TFE, TFB1, and TBP cDNAs was carried out on a Chromo-4 cycler with Maxima SYBR green Master Mix. DNA amplification was monitored in real time, and threshold cycle (Ct) values were obtained for each sample. TBP, TFB1, and TFE qPCRs were carried out in triplicate, and the standard error for each sample (error bars) was calculated. No-template and no-reverse-transcriptase negative controls were carried out in triplicate and duplicate, respectively, and showed no DNA amplification. Ct values were converted into fold change in TBP, TFB1, and TFE mRNA levels at different times and are shown in the table.