Camkkβ

The isolated NCI-H716 cells and duodenal tissues were lysed with cell lysis buffer. The isolated intestinal tissues were lysed with tissue lysis buffer (Thermo Fisher, United States) and immunoblotting was performed as described by Kim et al. (2016 (link), 2017b (link)) and Lee et al. (2017) (link). Individual proteins were detected with primary antibodies against the phosphorylated form of AMPK (p-AMPK, T172; 1:1000), total AMPK (t-AMPK; 1:1,000), CAMKKβ (1:1,000), and β-actin (1:1,000). The p-AMPK and AMPK were purchased from Cell Signaling (Danvers, MA, United States). CAMKKβ and β-actin were purchased from Santa Cruz Biotechnologies (CA, United States) (Jang et al., 2017 (link)).

The total proteins of the sciatic nerve tissues were extracted with a Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeonggi-Do, Korea), following the manufacturer’s instructions. Western blot assay was performed with specific antibodies for CaSR (Thermo Fisher Scientific Inc, Waltham, MA, USA), CaMKKβ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), total LKB1 (Cell Signaling Technology, Danvers, MA, USA), phosphor-Ser⁴²⁸ LKB1 (Cell Signaling Technology, Danvers, MA, USA), total AMPK (Cell Signaling Technology, Danvers, MA, USA), phospho-Thr¹⁷² AMPK (1:2000; Cell Signaling Technology, Danvers, MA, USA), total eNOS (Cell Signaling Technology, Danvers, MA, USA), phospho-Ser¹¹⁷⁷ eNOS (Cell Signaling Technology, Danvers, MA, USA), PGC-1α (Novus Biologicals, Littleton, CO, USA), B cell leukemia/lymphoma 2 (Bcl-2) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL-2-associated X protein (Bax) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), beclin-1 (Novus Biologicals, Littleton, CO), and LC-3 (Sigma-Aldrich, St. Louis, MO). After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling Technology, Danvers, MA), target proteins were visualized by an enhanced chemiluminescence substrate (ECL Plus, GE Healthcare Bio-Sciences, Piscataway, NJ).