Anti β actin

The specimens were cut and homogenized in RIPA Buffer with Protease Inhibitor Cocktail (Nacalai tesque, Kyoto, Japan). The homogenate was then centrifuged at 15,000 rpm for 10 min at 4 ˚C. We separated the supernatants of the homogenates using sodium dodecyl sulfate polyacrylamide gel electrophoresis (12.5% e-PAGEL-HR, ATTO) and transferred the proteins onto an Immobilon-P membrane (Clear Blot Membrane-P plus, ATTO). We blotted the membranes with anti-ERK1/2 (#9102, rabbit, polyclonal, 1:1000, Cell Signaling Technology), anti-pERK1/2 (#4370, rabbit, monoclonal, 1:500, Cell Signaling Technology), or anti-β-actin (G043, mouse, 1:1000, abm) antibodies. We treated the sections with a secondary antibody (EnVision+/HRP, Dual Link Rabbit/Mouse, HRP) and visualized the protein bands with a chemiluminescence detection system (Amersham ECL Prime Western Blotting Detection Reagent). We analyzed the signals in the immunoblots using an LAS-4000 digital imaging system (Fujifilm, Tokyo, Japan). We quantified and divided the ERK and p-ERK band intensities by their corresponding loading controls with Image J software (version 1.52u) and determined the ERK phosphorylation rate by p-ERK/ERK.

Colonoids were resuspended in RIPA buffer with protease inhibitors and phosphatase inhibitor, sonicated, then centrifuged at 16,000xg for 20 min at 4 °C. Total protein was estimated and 10 μg of whole cell lysate prepared according to manufacturer’s instructions in 1X Bolt LDS Sample Buffer with 1X Bolt Reducing Agent (Life Technologies) and heated at 70 °C for 10 min. Proteins were separated by Bolt 12% Bis–Tris Gel (Life Technologies), transferred to PVDF membrane (Life Technologies), followed by immunoblotting with rabbit monoclonal phospho-p38 (1:2000; Cell Signaling Technologies #9211), total p38 (1:2000; Cell Signaling Technologies #9212), phospho-NFκB (1:2000; Cell Signaling Technologies #3033), total NFκB (1:2000; Cell Signaling Technologies #8242) or mouse monoclonal anti-β-actin (1:2000; ABM # G043), then with horse α-rabbit IgG:HRP (1:2000; Cell Signaling Technologies #7076) or horse α-mouse IgG:HRP (1:2000; Cell Signaling Technologies #7076). Western blot images were taken using a ChemiDoc imaging system (Bio-Rad) and densitometry analysis of the obtained images were done using ImageJ 1.45S software (Wayne Rasband, NIH).

Pull-down assays were performed using streptavidin agarose beads (Pierce, Rockford, IL, USA). Briefly, HUVEC lysates were preincubated with anti-α_vβ₃ antibody (Abcam) or IgG (Santa Cruz Biotechnology) for 15 min at 4 °C with rotation and then incubated with biotinylated-OPNpt20 (bt-OPNpt20, 0.1 μg μl⁻¹) or biotinylated-OPNpt20-RAA (bt-OPNpt20-RAA, 0.1 μg μl⁻¹) for 30 min. These mixtures were incubated with 20 μl of streptavidin beads for 30 min at 4 °C, centrifuged at 8000 r.p.m. for 2 min, washed three times and analyzed by immunoblot using anti-integrin αV (1:1000; Santa Cruz Biotechnology) and anti-β-actin (1:4000; Applied Biological Materials) antibodies.