Anti phospho myosin light chain 2

For western blot analysis, crypts were isolated by the same method for colonoid culture described above. Crypts were pelleted by centrifugation and resuspended in RIPA buffer, phosphatase inhibitor cocktail 2 (Sigma, P5726), phosphatase inhibitor cocktail 3 (Sigma, P0044), protease inhibitor (Sigma, P8340), and 2X Laemmli buffer and then boiled for 5 minutes before analysis by SDS-PAGE electrophoresis. Membranes were blocked using Odyssey blocking buffer for 30 minutes and then blotted with anti E-cadherin (BD Transductions, #610182), anti-ZO-1 (Life Technologies, #617300), anti-Claudin7 (Invitrogen #QL221418), anti-GAPDH (Cell Signaling, #5174S), anti-β-actin (Sigma, #116M4801V), anti-myosin light chain 2 (Cell Signaling, #8505S), anti-phospho-myosin light chain 2(Cell Signaling, #3674S), or Myosin 2C (Cell Signaling, #8189S). All antibodies were used at 1:1000 concentration (except anti-β-actin, 1:2000) in Odyssey blocking buffer with 0.1% Tween-20 and incubated overnight at 4°C. LiCor secondary antibodies were used at 1:10,000 dilution and incubated for 2 hours at room temperature. Quantification of western blot band intensity was conducted using LiCor Image Studio.