FIG. 4.

Constitutively expressed Mot3 does not affect the rate of hypoxic gene induction. (A) RZ53-6Δmot3 cells containing Mot3-HA expressed from its native promoter (Mot3-HA, lanes 1 to 4) or constitutively expressed Mot3-HA (pT1-M3-HA, lanes 5 to 8) were grown aerobically (0) or anaerobically for the number of hours indicated (1, 2, and 4), and RZ53-6Δmot3 cells containing untagged Mot3 (UT, lane 9) were grown aerobically. Crude protein extracts were prepared and subjected to an immunoblot with monoclonal antibody against the HA epitope. Equal loading of the samples was determined by Ponceau S staining (data not shown). (B) Total cellular RNA was prepared from RZ53-6Δmot3 cells containing Mot3 expressed from its native promoter (Mot3-HA, lanes 1 to 6) or constitutive Mot3 (pT1-M3-HA, lanes 7 to 12) grown to mid-exponential phase aerobically (0) or anaerobically for the number of hours indicated (0.5, 1, 1.5, 2, and 4). RNA was hybridized with ³²P-labeled probes to ANB1, HEM13, and ACT1 (as a loading control). The positions of the specific RNAs are indicated to the left of the blot. While a probe to TIF51A was not used, its high degree of sequence similarity to ANB1 resulted in cross-hybridization.