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. 1997 Nov 11;94(23):12503–12508. doi: 10.1073/pnas.94.23.12503

Figure 3.

Figure 3

Nitrogen source does not affect protease-resistance of Ure2p. [ure-o] and [URE3] derivatives of strain 3389/p530 (MATa kar1 ura2 leu2 his− YEp351URE2) from Leu plates were grown to midlog phase in 2% dextrose with uracil and histidine and either ammonia (yeast nitrogen base; Difco) or proline (0.2% in yeast nitrogen base without ammonia; Difco) as nitrogen source. Cell lysates were prepared and treated as described (9). Reactions contain 2.5 mg/ml protein. After removing an aliquot as a PK control, proteinase K was added to the remainder to a final concentration of 45 μg/ml, and reactions were transferred to 37°. Aliquots were removed at indicated time points and transferred to sample buffer containing 3 mM phenylmethylsulfonyl fluoride and frozen on dry ice. Samples were run on 12% polyacrylamide/SDS gels, and immunoblotted with anti-Ure2p antibody. Molecular mass size markers are indicated. C = 15 μg of protein from the [URE3] strain, no treatment. 0 = PK; 1, 5, 10 = minutes of proteinase K digestion. [ure-o] means absence of the [URE3] genetic element.