Figure 2. Mutational analysis of Akt1 SUMO conjugation sites. (A) HEK 293T cells were transfected either with wild-type (WT), K276R, K301R, or the double mutant K276R/K301R (“2KR”) HA-tagged Akt1 expression vectors, together with 6xHis-tagged SUMO2 and Ubc9 expression vectors. Cells lysates were subject to denaturing Ni2+ affinity chromatography and analyzed by western blot with an anti-HA antibody as indicated in Figure 1. Different lanes from the same gel were put together as indicated by the dividing lines. (B) Analysis of the SUMO consensus site surrounding K276 in Akt1. CM, canonical consensus motif; ICM, inverted consensus motif; Ψ, bulky, hydrophobic amino acid; X, any amino acid; E, glutamic acid; D, aspartic acid. (C) HEK 293T cells were transfected either with wild-type, K276R, or the double mutant D274N/E278Q HA-tagged Akt1 expression vectors, together with 6xHis-tagged SUMO2 and Ubc9. Cell lysates were subject to denaturing Ni2+ affinity chromatography and analyzed by western blot with an anti-HA antibody as indicated above. Different lanes from the same gel were put together as indicated by the dividing line.