Anti ano1

Microscopic analysis of copGFP fluorescence within the jejunum and colon was performed as previously described [13 (link)]. Jejunal and colonic tissue was analyzed by whole mount and cryostat section staining using confocal microscopy as previously described [19 (link)]. Primary antibodies against the following antigens were used: anti-ANO1 (mouse, 1:200, Abcam, Cambridge, MA), anti-KIT (goat, 1:20, R&D Systems, MN), anti-THBS4 (rabbit, 1:100, Santa Cruz, CA), and anti-HCN4 (rabbit, 1:100, alomone labs, Jerusalem, Israel). Images were collected using the Fluoview FV10-ASW 3.1 Viewer software (Olympus, Tokyo, Japan) with an Olympus FV1000 confocal laser scanning microscope.

Protein samples (80 μg) were separated using 4%–12% Tris-Glycine-PAG Pre-Cast Gel (Koma Biotech, South Korea) and transferred to polyvinylidene fluoride membranes. Blocking was performed using 5% bovine serum albumin (BSA) in tris-buffered saline with 0.1% Tween 20 for 1 h. The membranes were then incubated with the corresponding primary antibodies, including anti-ANO1 (Abcam, United Kingdom) and anti-β-actin (Santa Cruz Biotechnology, United States), followed by incubation with horseradish peroxidase-conjugated anti-secondary IgG antibodies (Enzo Life Science) for 1 h. Finally, visualization was performed using the ECL Plus Western Blotting System (GE Healthcare).

Cell lysates were analyzed by SDS-PAGE and western blotting using standard protocols and the following primary antibodies: anti-ANO1 (Abcam, SP31), anti-EGFR (Cell Signaling), anti-Tubulin (Sigma), anti-FLAG (Sigma-Aldrich). Blots were analyzed using an Odyssey scanner (LICOR).