α7nachr

Colon proteins were extracted as described previously (25 (link)). Briefly, RIPA buffer supplemented with protease inhibitors (Sangon Biotech Co., Ltd.) was used as lysis buffer. Colon proteins were separated by 10% SDS-PAGE, and the gels were then electro-transferred onto nitrocellulose filter membranes (Whatman; Cytiva). The membranes were incubated with antibodies against α7nAChR (Sigma-Aldrich; Merck KGaA, cat. no. M220, 1:1,000), phosphorylated (p-)SHP2 (Abcam; cat. no. ab62322, 1:500), SHP2 (Abcam, cat. no. ab131541, 1:500), p-STAT3 (Cell Signaling Technology, Inc.; cat. no. 9145, 1:1,000); STAT3 (Santa Cruz Biotechnology, Inc., cat. no. sc-482, 1:100), p-Jak2 (Abcam, cat. no. ab32101, 1:1,000); Jak2 (cat. no. ab108596, Abcam, 1:1,000) or GAPDH (Abcam, cat. no. ab181602, 1:2,000) overnight at 4°C. The membranes were then incubated with an IRDye 800CW-conjugated secondary antibody (Rockland Immunochemicals Inc. 1:20,000) for 1 h at room temperature. Images were acquired using an Odyssey infrared imaging system (Odyssey^® CLx Imaging System, LI-COR Biosciences) and ImageJ software v1.52 (NIH) was used for analysis.

Equal amounts of total protein from extracts of thigh muscle and HUVECs were resolved on SDS 10% PAGE and transferred to nitrocellulose membranes for Western blotting with HIF‐1α (1:200 dilution), phospho‐VEGFR2 (p‐VEGFR2; Tyr996, 1:100 dilution), phosphor‐protein kinase B (p‐Akt; Ser473, 1:100 dilution), and β‐actin (1:200 dilution; Cell Signaling Technology), as well as with choline acetyltransferase (ChAT; 1:100 dilution), vesicular acetylcholine transporter (VAChT; 1:100 dilution), and α7‐nAChR (1:200 dilution; Sigma‐Aldrich), followed by incubation with HRP‐conjugated secondary Ab (GE Healthcare, Fairfield, CT), as described previously.^{16 (link)} Positive signals were detected with an enhanced chemiluminescence (ECL) system (GE Healthcare). Relative protein levels were quantified using an ECL Western blotting detection kit (GE Healthcare) and ImageJ software (National Institutes of Health, Bethesda, MD).