Figure 7.

Ser25 phosphorylation appears to trigger the formation of high‐molecular‐mass forms of AnxA2. Recombinant wt His‐AnxA2 (lane 1, 1 μg) and the phospho‐mimicking His‐AnxA2‐Ser25Glu (lane 2, 1 μg) were bound to Ni²⁺‐resin (lanes 4 and 5 respectively) and incubated with PC12 total cell lysate for 1 h before elution with 250 imidazole buffer. 1.5 μg of the eluted wt His‐AnxA2 (lane 4) and His‐AnxA2‐Ser25Glu (lane 5) were loaded on the gel. As a control, elution of proteins present in the PC12 cell lysate that may bind unspecifically to the resin was also performed (lane 3). Proteins were separated by 10% SDS/PAGE and subjected to western blot analysis. The blots were probed with antibodies against AnxA2. Detection of the resulting protein bands was performed using the ChemiDoc™ XRS+ molecular imager after incubation with HRP‐conjugated secondary antibodies and ECL reagent. The arrowheads to the left indicate the protein molecular mass standards.