Fig. 8

Pull-down and Dot-blot assay of the recombinant proteins with a known protein interactor, FAP174. a All four recombinant proteins purified to homogeneity were checked for their bioactivity by performing a pull-down assay with a known interactor. a–c Controls with GST alone binding to Glutathione-Sepharose beads, GST bound to the beads followed by addition of purified FAP174 (no binding seen when beads are electrophoresed on an SDS-PAGE gel, see lane marked Bd (for beads) and FAP174 bound to beads followed by FT (flow-through) and washes (W). Note the absence of FAP174 protein in b and c. d, e The CrFAP65AH1 (H1 and its variant (H1v) pure proteins were individually used in the pull-down assay. FAP174 along with CrFAP65AH1 (lane marked Bd in d) was seen, indicating a direct interaction. The variant, on the other hand, was not pulled down by FAP174 (see lane marked Bd in e). FAP174 was found to be present in the flow-through (lane marked FT in e). f, g The CrFAP65AH2 (H2 and its variant (H2v) pure proteins were individually used in the pull-down assay. FAP174 along with a very low amount of CrFAP65AH2 (lane marked Bd in f) was seen, indicating a direct, but weak interaction. The variant, on the other hand, was not pulled down by FAP174 (see lane marked Bd in g). Note the presence of FAP174 in the flow-through (lane marked FT in g). The brightness and contrast of all the images has been adjusted to 15%. h Dot blot followed by overlay assay indicated that CrFAP65AH1 and CrFAP65AH2 binds to FAP174 whereas no binding is observed in CrFAP65AH1V12P, CrFAP65AH2V12P, GST, Primary antibody control, Secondary antibody control