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. 2008 Dec 3;296(2):C285–C295. doi: 10.1152/ajpcell.00418.2008

Fig. 2.

Fig. 2.

GST-KCa3.1 pull-down assay. A: Coomassie blue-stained SDS-PAGE gel of purified GST (lane 1) and GST-KCa3.1 terminal fusion protein (lane 2) confirming the purity of GST and the GST KCa3.1. B: Western blot obtained using a rabbit anti-KCa3.1 antibody raised against a synthetic peptide corresponding to amino acid residues 350–363 of rat KCa3.1 (1:1,000, Sigma-Aldrich). C: evidence for 5′-AMP-activated protein kinase γ1-subunit (AMPK-γ1) being pulled down by the GST-KCa3.1 fusion protein. Western blots obtained using a rabbit anti-AMPK-γ1 antibody (1:2,000) with horseradish peroxidase-conjugated goat anti-rabbit IgG as secondary antibody. HIK lysate (lane 1C), obtained as described in materials and methods, was added to confirm that endogenous γ1-subunit of AMPK migrates at the same MW than the AMPK-γ1 from pulldown. Western blot also demonstrates that the GST-KCa3.1 fusion protein is able to bind to the γ1-subunit of AMPK, whereas no binding is detected with GST alone.