Figure 6

Nme1/NmeGp1Sd complex formation analysis: A) Input control: Cell lysates from control K0 (CAL 27 cells stably transfected with pcDNA3 vector), K1 and K2 (CAL 27 cells stably transfected with pcDNA3FLAG/Nme1 construct) and S1 and S2 (CAL 27 cells stably transfected with pcDNA3FLAG/NmeGp1Sd constructs) tested with anti-FLAG antibody. B) Cell lysates from control K0 (CAL 27 cells stably transfected with pcDNA3 vector), K1 and K2 (CAL 27 cells stably transfected with pcDNA3FLAG/Nme1 constructs) and S1 and S2 (CAL 27 cells stably transfected with pcDNA3FLAG/NmeGp1Sd constructs) tested with anti-Nme1 antibody. C) Immunoprecipitation: FLAG/Nme1 (K1 and K2) and FLAG/NmeGp1Sd (S1 and S2) were immunoprecipitated with anti-FLAG M2 affinity gel and immunoblotted with anti-Nme1 antibody. K0-control (clone with "empty" construct). FLAG/Nme1 produces heteromers with exogenous (upper band) and endogenous (lower band) Nme1. FLAG/NmeGp1Sd produces complexes with the endogenous (human) Nme1 while the upper band (exogenous, FLAG/NmeGp1Sd) cannot be seen, since the antibody is specific for human Nme1. D) Immunoprecipitation: FLAG/Nme1 (K1 and K2) and FLAG/NmeGp1Sd (S1 and S2) were immunoprecipitated with anti-FLAG M2 affinity gel and immunoblotted with anti-FLAG and anti-Nme1 antibody. K0-control (clone with "empty" construct). FLAG/Nme1 produces heteromers with exogenous (upper band) and endogenous (lower band) Nme1. FLAG/NmeGp1Sd produces complexes with the endogenous (human) Nme1. The upper band (exogenous, FLAG/NmeGp1Sd) is visible, since it is stained with anti-FLAG antibody.