Figure 3.

Northern blot hybridization, RNase protection assays, and Western blot analysis of the Psr1 protein. (A) Northern blot hybridization of a Psr1 gene-specific probe to RNA from the wild-type strain CC125. Polyadenylated RNA from cells grown in nutrient-replete medium (+P_i) and from cells starved for 8–48 h and pooled (−P_i) was analyzed. (B) RNase protection of a 395-base riboprobe by 30 μg of total RNA. The level of accumulation of the transcript (fold change relative to +P_i conditions) is indicated beneath each lane. This experiment was repeated on separately isolated RNA samples, and the trend was identical. (C) Western blot analysis of the Psr1 protein containing the 3×HA tag. The first two lanes contain protein that was extracted from cells in which Psr1 did not contain the 3×HA tag. The second two lanes contain protein that was extracted from the psr1–1 mutant containing the PSR1∷3×HA construct. Protein extracts were from either cells grown in complete medium (+P_i) or cells starved for P for 24 h (−P_i). Three independent transformants were examined, and all three gave essentially identical results. To control for loading, the blot was stripped and reprobed for D1 protein (32-kDa polypeptide), which was shown previously not to change in abundance after 24 h of phosphorus starvation (6).