Protein Extraction and Western Blotting from Colonic Tissues

As previously described (Prigent et al., 2019 (link)), protein extraction from colonic tissues was performed with NucleoSpin RNA/Protein Kit (Macherey-Nagel, Hoerdt, France, Cat# 740966) according to the manufacturer instructions. Samples were further prepared for electrophoresis by diluting with a NuPAGE sample buffer (Life Technologies, Saint-Aubin, France, Cat# NP0008) then heated at 98°C for 5 min. Lysates were separated using the NuPAGE 4–12% Bis-Tris gels (Life Technologies, Cat# NP0336BOX) together with the 2-(N-morpholino)ethanesulfonic acid/sodium dodecyl sulfate running buffer (Life Technologies, Cat# IB23002) before electrophoretic transfer to nitrocellulose membranes (Life Technologies, Cat# NP0002) with the iBlot2 Dry Blotting System (Life Technologies, Cat# IB21001). Membranes were then blocked for 1 h at 21°C in Tris-buffered saline (Sigma, Cat# T5912) with 0.1% (v/v) Tween-20 (Sigma, Cat# P1379) and 5% (w/v) non-fat dry milk and incubated overnight at 4°C with the following primary antibodies: rabbit anti-GR (D8H2, 3660S 1:500, Cell Signaling, Danvers, MA, USA), mouse monoclonal anti-β-actin (1:10000; Sigma, Cat#A5441, RRID:AB_476744). Bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit (Life Technologies Cat# 31460, diluted 1:5000) or anti-mouse antibodies (Sigma, Cat# A9044, diluted 1:5000) and visualized by enhanced chemiluminescent detection (Biorad, Clarity ECL, Marnes-la-Coquette, France, Cat# 170-5061).