In order to obtain additional confirmation for the existence of the complexes deduced from the pull-down experiments described above, the eluates were further analyzed using non-denaturing conditions. To this aim, the immunoblot analysis was repeated after the proteins eluted from StrepTactin Opaganib datasheet columns were resolved in 4-20% gradient polyacrylamide

native gels (Figure  4, lower panels). When the immunoblot was developed with anti-HupL antiserum, a major immunoreactive band was detected in eluates from the ΔhupD derivative strain (Figure  4A). A band of similar size and mobility was detected when a replicate immunoblot was developed with the StrepTactin-AP conjugate (Figure  4B), suggesting that both bands correspond to a HupL-HupF SRT1720 datasheet complex. In both cases, the absence of HupK was associated to the virtual absence of

HupFST-containing complexes (Figure  4A and 4B). Finally, a third replicate of the same immunoblot developed with the anti-HupK antiserum revealed a fainter band, with a slightly lower mobility (Figure  4C), suggesting a different, less abundant HupK-HupF complex. As before, non-specific bands were detected by this antiserum in the ΔhupK mutant, likely corresponding to complexes of the non-specific bands detected in the SDS-PAGE experiments described above. Further confirmation on the composition of the complex or complexes detected by immunoblotting was sought by peptide mass fingerprinting analysis of the major complex present in the eluate obtained from the ΔhupD strain UPM 1155(pALPF4, pPM501). Such eluate was resolved by 4-20% gradient native PAGE, followed by Coomassie Blue staining. In this gel we identified a clear band with a mobility similar to that of the complexes identified above (data not shown). This band was excised and subjected to MALDI-TOF analysis after trypsin digestion. The analysis led to the identification of peptides corresponding to proteins HupL and HupF (data not shown), indicating the presence of a major

complex involving these two proteins. In this analysis no peptides corresponding to HupK, nor to any other Hup/Hyp proteins, were detected. Taken together, medroxyprogesterone data from immunoblot and mass spectrometry analyses suggest the presence of two different complexes: a major complex containing HupF and HupL, and a second, much less abundant complex involving HupF and HupK, only detectable through immunoblot analysis. Functional analysis of the HupF C-terminal region A distinctive domain of R. leguminosarum HupF is the extended C-terminal region, absent in the otherwise structurally related HypC protein (Figure  1). In order to elucidate the relevance of this region for HupF function, we constructed plasmid pPM501C, a pPM501 derivative in which the hupF gene was modified to produce a truncated version of HupFST (HupFCST) with a precise deletion of the C-terminal 24 amino acid residues of HupF (see Methods).