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. 2004 Apr;3(2):348–358. doi: 10.1128/EC.3.2.348-358.2004

FIG. 4.

FIG. 4.

The Δmak-2 mutant lacks a protein that is recognized by anti-p44/42 and anti-phospho p44/42 antibodies. (A) Total protein (30 μg) was extracted from wild-type strains (RLM 40-27 and Xa-2), Δmak-2 (PB-1), Δmak-2 progeny (APJ-1, APJ-3, and APJ-4), and nrc-1 and separated on an SDS-10% polyacrylamide gel electrophoresis gel. The blot was probed with anti-p44/42 (α-p44/42) antibodies (PhosphoPlus antibody kit; Cell Signaling Technology). Lane 1, nrc-1; lane 2, APJ-4; lane 3, APJ-3; lane 4, APJ-1; lane 5, RLM 40-27; lane 6, Xa-2; lane 7, PB-1. The lower panel shows blots probed with anti-β-tubulin antibodies for protein loading controls. (B) The introduction of mak-2 into Δmak-2 progeny APJ-1 restores the production of a protein that is recognized by anti-p44/42 antibodies. Total protein (30 μg) was extracted from APJ-1 transformants containing pOKEmak-2 (4C, 6C, and 7C), the wild type (RLM 47-20), and PB-1 Δmak-2 mycelia and separated by SDS-10% polyacrylamide gel electrophoresis followed by transfer onto a nitrocellulose membrane. Anti-p44/42 antibody (Cell Signaling Technology) was used for probing the blot. Lane 1, RLM 40-27; lane 2, Δmak-2 (PB-1); lane 3, APJ-1 transformant (4C) containing pOKEmak-2; lane 4, APJ-1 transformant (6C) containing pOKEmak-2; lane 5, APJ-1 transformant (7C) containing pOKEmak-2. (C) The Δmak-2 mutants lack a protein that is recognized by anti-phospho p44/42 antibodies. Total protein (30 μg) was extracted from the wild-type strain (RLM 40-27), Δmak-2 progeny (APJ-1, APJ-2, and APJ-4) and nrc-1 grown for 20 h as described in Materials and Methods and separated on an SDS-10% polyacrylamide gel electrophoresis gel. The blot was probed with anti-phospho p44/42 antibodies (PhosphoPlus antibody kit; Cell Signaling Technology). Lane 1, nrc-1; lane 2, APJ-1; lane 3, APJ-4; lane 4, APJ-2; lane 5, RLM 40-27. (D) The APJ-1 transformant (6C) containing mak-2 shows restoration of hyphal fusion. Conidia from the APJ-1 transformant containing pOKEmak-2 (6C) were inoculated onto cellophane layered onto plates containing Vogel's minimal medium. Colonies were assessed for hyphal fusion events after 24 h of growth. Arrows indicate hyphal fusion events. Bar, 10 μm.