FIG. 4.

Molecular analysis of cgl2 silencing in C. cinerea strain AmutBmut. Individual transformants with cgl2-silencing construct 359 (lanes 1 to 21) were analyzed and compared to mock transformants carrying the vector control 336 (lanes C1 to C6). (A) Analysis of DNA integration by Southern blotting. Genomic DNA of the individual transformants was analyzed by using the restriction enzyme HindIII and a cgl2 hybridization probe (see Materials and Methods for details). The positions of the endogenous cgl2 and the introduced cgl2hp fragments are indicated. A HindIII digest of plasmid 359 served as a positive control. DNA-Molecular-Weight-Marker VII (Roche) was used as size standard (m). (B) Analysis of CGL1/2 and CGL3 protein levels in WCEs prepared from fruiting mycelium and primordia of the individual transformants (see Materials and Methods for details). A total of 5 μg of WCE from the indicated sample of the indicated transformants (numbering refers to panel A) was analyzed by immunoblotting with specific antisera against CGL1/2 and related protein CGL3 as described in Materials and Methods. In case of the anti-CGL1/2 blots, the HRP-coupled secondary antibody was detected either by using ECL (fruiting mycelium) or by using less sensitive chloronaphthol development (primordia). The positions of the individual proteins are indicated.