Confirmation of AMPK-CFTR interaction by GST pull-down assays. (a) CFTR COOH-terminal tail (GST-CFTR-1411–1480) pulls down α1-AMPK in vitro. Purified liver AMPK holoenzyme (15 ng) was loaded in first lane as reference. (b) GST-α1-AMPK fusion protein expressed in CHO-BQ2 cells pulls down full-length CFTR in vivo. Ten micrograms of the total soluble cellular protein was loaded in first and third (lysate) lanes as reference for CFTR. Second and fourth (beads) lanes show eluate from GSH-Sepharose beads after affinity purification and washing. (c) Comparison of binding strengths of CFTR with NH₂-terminal and COOH-terminal GST-α1-AMPK fusion proteins in CHO-BQ2 cells. Lower panel is the same membrane probed with anti-GST antibodies to allow comparisons of different CFTR bands in panel above. (d) GST-α2-AMPK fusion protein expressed in CHO-BQ2 cells pulls down CFTR in vivo. All results shown are representative of at least three replicate experiments.