Figure 5.

AtMTAN forms a complex with CBL3 only in the presence of calcium in vitro. A, Purification of the recombinant AtMTAN protein expressed in E. coli. Lanes 1 to 3 contain the GST-AtMTAN fusion protein, the thrombin-digested forms of GST and AtMTAN, and purified AtMTAN, respectively. B, Pull-down assay. The GST-AtMTAN fusion protein was used as a bait to pull down the prey CBL3 in the presence (+) or absence (−) of calcium. For a negative control, GST was used as bait. The top panel shows an immunoblot probed with rabbit anti-CBL3 antibody, and the bottom panel shows a Coomassie Brilliant Blue-stained SDS-PAGE gel indicating the amount of bait proteins used in each pull-down assay. C, Affinity purification of CBL3 by GST-AtMTAN from the Arabidopsis flower total protein extract. Proteins retrieved from the plant extract by GST-AtMTAN and GST (control) affinity beads were analyzed with immunoblot assay using the anti-CBL3 antibody as a probe (top). Total protein extract (10 μg) from the Arabidopsis flowers was loaded in the first lane to show the size of the CBL3 protein. The bottom panel shows the immunoblot stained with Ponceau S indicating the amount of bait proteins used as affinity reagents. [See online article for color version of this figure.]