Chaps buffer

Whole abdominal wall samples (n = 30) were decellularized using an already published protocol validated previously in our lab [40 (link)]. Whole rat abdominal wall samples were incubated with decellularization buffers under vigorous agitation at 150 rpm. The decellularization procedure involved the incubation of the samples in CHAPS buffer (pH 7; 8 mM CHAPS, 1 M NaCl, and 25 mM EDTA in PBS 1× (Sigma-Aldrich, Darmstadt, Germany) for 18 h at room temperature (RT). Next, the rat abdominal wall samples were washed 3 times (10 min each time) with PBS 1× (Sigma-Aldrich, Darmstadt, Germany) under continuous agitation (150 rpm) at RT. The samples were incubated in SDS buffer (pH 7; 1.8 mM SDS, 1 M NaCl, and 25 mM EDTA in PBS 1×; Sigma-Aldrich, Darmstadt, Germany) for another 18h at RT. The samples were again washed 3 times (10 min each time) with PBS 1× (Sigma-Aldrich, Darmstadt, Germany) under continuous agitation (150 rpm) at RT. Finally, the samples were placed in a solution containing a-Minimum Essentials Medium (a-MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 40% v/v fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 36 h at 37 °C and washed again, as described before. The whole decellularization procedure was repeated another 2 times (in total, 3 decellularization cycles, n = 5 samples/decellularization cycle).

rES (n = 10, l = 4 ± 1 cm) were cut into 3 segments of 1 cm and submitted to decellularization buffers. The decellularization process was performed according to previous described protocols with some modifications [12 (link),13 (link)]. Briefly the esophagus segments were subjected to CHAPS buffer at pH 7 (8 mM CHAPS, 1 M NaCl and 25 mM EDTA in PBS 1X, Sigma-Aldrich, Darmstadt, Germany) for 6 h at room temperature under constant agitation at 350 rpm. Then, the esophageal segments were subjected to SDS buffer at pH 7, (1.8 mM SDS, 1M NaCl and 25 mM EDTA in PBS 1X, Sigma-Aldrich, Darmstadt, Germany) for additional 6 h at room temperature under constant agitation at 350 rpm. Finally, the esophageal segments were incubated in α-Μinimum Essentials Medium (α-ΜΕM, Sigma-Aldrich, Darmstadt, Germany) supplemented with 40% v/v Fetal Bovine Serum (FBS, Sigma-Aldrich, Darmstadt, Germany) for 6 h at 37 °C under constant agitation at 350 rpm. The above procedure was repeated for 1 more cycle.

Cells were lysed in CHAPS buffer (Sigma-Aldrich) containing the protease inhibitor cocktail V (Calbiochem, Billerica, MA) and the phosphatase inhibitor cocktail II (Sigma-Aldrich). HER3 immunoprecipitation was performed by incubating 2 mg of cell lysate with 2 μg of the anti-HER3 antibody 2F12, which recognizes the HER3 intracellular C-terminal tail, at 4°C for 6 hr, followed by overnight incubation with 20 μl of protein A/G agarose beads (Santa Cruz Biotechnology) or with magnetic beads (Dynabeads^™; Life Technologies) at 4°C under agitation. Samples were washed five times with 400 μl CHAPS buffer, re-suspended in 100 μl of 2X SDS Laemmli buffer and heated at 90°C for 10 min before electrophoresis. In each case, no HER3 protein was immunoprecipitated with beads alone or with normal control IgG antibody.