Recombinant human BMP-6, BMP-9, VEGF, bFGF and TGF-β were
purchased from R&D Systems (R&D Systems, Bio-Techne, Minneapolis,
MN, USA). Cycloheximide (CHX) was purchased from Sigma-Aldrich (Merck,
Darmstadt, Germany), and MG132 was from Peptide Institute (Osaka, Japan). The
following antibodies were used: anti-FLAG M2 (F3165; Sigma-Aldrich, Merck),
anti-DDDDK (PM020; MBL, Tokyo, Japan) which recognizes the FLAG epitope,
anti-Myc, anti-HA (HA124; Nacalai Tesque, Kyoto, Japan), anti-HA (#561; MBL),
anti-α-tubulin (AC-15; Sigma-Aldrich, Merck), anti-SMAD1 (913C1b; Bio
Matrix Research, Chiba, Japan) which recognizes both SMAD1 and SMAD5 for ChIP
assays (5 (link)), anti-phospho-SMAD1/5/8 (Cell
Signaling Technology, Danvers, MA, USA), anti-PECAM1 (clone 18; BD bioscience,
San Jose, CA, USA), anti-HIF-1α (NB100-449, Novus Biologicals,
Bio-Techne), anti-HIF-2α (NB100-122, Novus Biologicals, Bio-Techne),
anti-α-smooth muscle actin (clone 1A4; Sigma-Aldrich, Merck), and
anti-HDAC1 (clone 2E10, #05-100; Millipore, Merck). A rabbit polyclonal
anti-ATOH8 antibody was raised against residues 306-321 of human ATOH8,
CTRTLQAEGRAKKRKE.