The NifA protein levels are higher in H. seropedicae strains lacking both Fnr1 and Fnr3. Protein extracts from different H. seropedicae strains were prepared and then hybridized with ANTI-FLAG antibodies as described in Methods. (A) Western hybridization of protein samples after resolution on 12% SDS-PAGE and transfer to PVDF membrane. (B) Protein loading control SDS-PAGE of samples used for hybridization in A. Lanes are as follows: 1, Protein molecular weight standard (MW); 2, H. seropedicae MBF1 (fnr1-3xFlag); 3, H. seropedicae MBN4 (nifA-3xFlag); 4, H. seropedicae MBN5 (nifA-3xFlag in the double fnr1, fnr3 deletion background); 5, H. seropedicae MBN6 (nifA-3XFlag in the triple fnr mutant background); 6, H. seropedicae SmR1 (no Flag protein control). In A, the MW used was Precision Plus Protein WesternC™ Pack (BioRad #161-0385), while in B, the MW used was the LMW-SDS Marker Kit (GE healthcare# 17-0446-01). Thin arrows indicate the molecular weight in KDa of protein standards. The black thick arrow indicates the NifA-3xFlag protein (~64 KDa), while the light grey thick arrow indicates the Fnr1-3xFlag protein (~34K Da). A representative result is show from three independent protein extract preparations.