Figure 3.

AtCBL1 is a functional Ca²⁺-binding protein. In the gel-shift panel, lane 1 and lane 2 were loaded with protein extract prepared before and after isopropyl β-d-thiogalactopyranoside (IPTG) induction, respectively. GST-AtCBL1 fusion protein was purified and incubated in EGTA- (lane 3) or calcium-containing (lane 4) buffer before being analyzed by SDS/PAGE. As a negative control, GST protein was analyzed in the same way (lanes 5 and 6). In the overlay assay, lanes 1–3 show Coomassie blue-stained molecular weight marker, GST-AtCBL1 (0.4 μg), and GST (0.2 μg), respectively. Lanes 4–6 indicate the same samples transferred to the nitrocellulose membrane and assayed by ⁴⁵Ca²⁺ binding. Only the fusion protein in lane 2 shows Ca²⁺-binding capability (lane 5).