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. 2006 Sep 29;188(23):8222–8230. doi: 10.1128/JB.00747-06

FIG. 3.

FIG. 3.

Immunoblot detection of mutant and wild-type GALLS proteins and Cre::GALLS fusion proteins. Numbers beside each panel indicate molecular weight standards (in thousands). Panel A shows an immunoblot probed with polyclonal rabbit antibodies raised against the purified GALLS protein. Soluble proteins were extracted from A. tumefaciens MX358 expressing the following: no GALLS protein (vector-only control; lane 1), wild-type GALLS (lane 2), GALLSΔ273-282 (helicase motif III deletion; lane 3), GALLS-K172E (Walker A mutation; lane 4), GALLS-D239N (Walker B mutation, lane 5), wild-type GALLS (lane 6), GALLSΔ705-723 (NLS deletion, lane 7), or GALLS plus TEV NLS (lane 8). Protein samples in lanes 2 to 5 were extracted from strains that contain the GALLS gene in a multicopy IncP plasmid (pVK100), whereas samples in lanes 6 to 8 were extracted from strains that contain the GALLS gene in a single-copy replicon based on an Ri plasmid ori (pDM12), which may explain the lower levels of the GALLS protein in these samples. Panel B shows an immunoblot probed with Cre-specific antibodies. Soluble proteins were extracted from A. tumefaciens LBA1100 expressing the following: Cre::GALLS-27R>Dx2 (lane 1), Cre::GALLS-27ΔR (lane 2), Cre::GALLS-27Δ6 (lane 3), Cre::GALLS-27Δ2 (lane 4), Cre::GALLS-27R>E/E>R (lane 5), Cre::GALLS-11 (lane 6), Cre::GALLS-27 (lane 7), or Cre (lane 8). Cre::GALLS-27 protein levels in A. tumefaciens LBA2587 were comparable to those in LBA1100 (data not shown).