Each MEKK1 mutant was overexpressed in 293T cells in the highly expressing pApuro vector and analyzed via western blot to check for protein expression.
The inhibition of the RACK1-p19 interaction is not due to a mis-targeting to different compartment, since we detected the same p19 and RACK1 perinuclear staining when the W164A mutant was overexpressed (our unpublished results).
When this mutant is overexpressed and its interaction with Ubiquitin examined by using STINT-NMR, the resulting spectra (Fig. S2) and surface interaction maps (Fig. S4) for both the unphosphorylated and phosphorylated states are largely identical to those obtained using unphosphorylated wild-type STAM2.
The I260Q mutant was overexpressed and purified in the same manner as wild-type (WT) Pol β (28).
Limited functional studies were conducted wherein human ETNK1 protein (wild-type and N244S mutant) was overexpressed in murine Ba/F3 cells by means of a murine stem cell virus retroviral expression vector.
The ErbB2 mutant was overexpressed in the cells after gene transfection, but PEPD failed to stimulate its phosphorylation, whereas under the same experimental condition, PEPD stimulated the phosphorylation of the wild-type ErbB2.
Since Lodish's group has shown that the constitutively active V617F mutant of JAK2 does not manifest its activity unless the EPO receptor is present [ 65, 66] (or if the mutant is overexpressed [ 67]), the activation process is likely a trans-mediated activation event within a receptor/JAK2 dimer rather than a cis-activation process.
As mentioned above, the A212P mutant may function as a dominant negative by interacting with seipin-binding partners through its cytosolic N- or C-termini, thus sequester and prevent the binding partners from interacting with endogenous wild-type seipin when A212P mutant is overexpressed.
HbpR and HbpR mutants were overexpressed and purified from E. coli.
