Fig. 3

NME6 proteins lack NDP kinase activity and exhibit preferentially the monomeric state. a Recombinant NME6 proteins lack NDP kinase activity. Purified NME1-His (positive control), NME6-186-His and NME6-194-His recombinant proteins were subjected to an enzymatic activity assay where the decrease of NADH absorbance at 340 nm over time is directly proportional to NDP kinase activity (n = 6). b Endogenous NME6 lacks histidine phosphorylation. MDA-MB-231T cell lysate was immunoblotted with and without preserving histidine phosphorylation, using anti-NME1, a mix of anti-phospho-histidine 1-pHis and 3-pHis and anti-NME6 antibodies. Note: A single overlapping signal is observed only in preserving conditions between NME1 and pHis (turquoise), corresponding to pHis-NME1 (positive control). c NME6 recombinant proteins remain mostly monomeric under crosslinking conditions. Glutaraldehyde crosslinking with pure recombinant NME6-186-His or NME6-194-His proteins was analyzed by Western blotting and protein staining with naphthol blue. d NME6 recombinant proteins are mostly monomeric. Size exclusion chromatography was performed by loading purified recombinant proteins NME6-186-His and NME6-194-His onto a Superdex 200 Increase 10/300 GL column, calibrated for molecular mass by a series of standard proteins. The protein elution profile was recorded by absorbance at 215 nm. The elution volume of monomers (M), dimers (D), trimers (T) and hexamers (H) expected from the calibration is indicated by arrows