Fig. 5. Increased calcium concentration causes increased association between AIP1 and PYK-2. Increased intracellular Ca²⁺ stimulates PYK-2 phosphorylation prompting an investigation of whether increasing the level of free calcium enhanced the AIP1-PYK-2 interaction. PYK-2 immunoprecipitations from transiently AIP1-transfected HEK293 cells were performed in various immunoprecipitation buffers. The IP buffer (control; containing 1 mM EGTA) was supplemented with 1 mM (lane 2) or 10 mM (lane 3) CaCl₂; external Ca²⁺ was complexed by raising the EGTA concentration to 5 mM (lane 4), or protein-associated Ca²⁺ was complexed by adding 5 mM BAPTA (lane 5). No impact on the AIP1-PYK-2 or actin-PYK-2 interaction was found after moderate increases in the CaCl₂ concentration, nor after addition of EGTA or BAPTA. However, raising the CaCl₂ concentration to 10 mM resulted in a dramatic increase of AIP1 and actin in PYK-2 immunoprecipitates (lane 3). PYK-2 activity, as measured by phosphotyrosine western blot, is elevated in the presence of 10 mM CaCl₂. The AIP1 band appears weaker in lanes 1, 2, 4 and 5 of this figure compared with that in Fig. 4 because of the short exposure time of the film to the blot, as a result of the strong signal in lane 3.