Increased apoptosis and reduced α-SMA staining were observed with complete freeing of the tissues from the posts but not upon removal of the magnet, resulting in a twofold decrease in post stiffness.
Proteinase K was added to final concentration of 200 ng mL−1, incubated for 2 h and supplemented with complete (EDTA free) protease inhibitors cocktail (Cat#1836170; Roche, Basel, Switzerland).
Loose and firm mucus from the in vivo measurements was collected by suctioning (loose) or gentle scraping (firm) in PBS supplemented with Complete EDTA-free protease inhibitor (Roche, Basel, Switzerland) and the samples were frozen.
The cell pellet was resuspended in lysis buffer [50 mM HEPES pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM TCEP, 10 mM imidazole] supplemented with cOmplete, EDTA-free protease inhibitor mix (Roche) and cells were lysed by a freeze thaw cycle in liquid nitrogen.
Lysis buffer was supplemented with Complete EDTA-free protease inhibitor cocktail (Roche).
Total mucus from the distal colon of DSS treated animals was removed by gentle scraping in PBS supplemented with Complete EDTA-free protease inhibitor (Roche, Basel, Switzerland).
2×106 B cells were lysed in 50 µl lysis buffer (150 mM NaCl, 50 mM Tris pH 7,4, 1% CHAPS) supplemented with Complete EDTA-free Protease inhibitor (Roche, Mannheim, Germany).
Twenty-four hours following transfection, cells were lysed with 1× passive lysis buffer (Promega) supplemented with complete EDTA-free protease inhibitors.
The treated cells were also lysed in 1 × cell lysis buffer (Beyotime) with complete EDTA-free protease inhibitor (Roche).
AVICs in 6-well plates were lysed in 1 × cell lysis buffer (Beyotime) with complete EDTA-free protease inhibitor (Roche).
