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. 2016 Aug 12;291(39):20588–20601. doi: 10.1074/jbc.M116.740191

FIGURE 2.

FIGURE 2.

Analysis of the role of GDH2 in the utilization of amino acids as the sole source of carbon. A, strategy for the generation of Δgdh2 strain. The restriction enzymes used for the digestion of genomic DNA for use in Southern analysis are indicated. P1, P2 primer pairs used in PCRs are indicated. B, PCR and Southern blotting analysis of genomic DNA isolated from GS115 (1) and Δgdh2 (2) strains. The Zeocin expression cassette is amplified by PCR only in Δgdh2 strain. M, DNA molecular weight markers (kb). The 4.4- and 2.5-kb bands hybridize to a radiolabeled probe (−571 to −24 bp of GDH2) in the Southern blot as expected. C, analysis of growth of GS115, Δmxr1, and Δgdh2 strains cultured in media containing different carbon sources. D, Western blotting analysis of Δmxr1 overexpressing Mxr1p from GAPDH promoter as Myc-tagged protein and Δgdh2 expressing Gdh2p from its own promoter as His-tagged protein. Anti-Myc and anti-His tag antibodies were used to detect Mxr1p and Gdh2p, respectively. E, quantitation of data presented in D. F, analysis of growth of different P. pastoris strains in various media as indicated.