Hoxd3

Tissue sections were fixed, processed, and stained as previously described [28 (link)]. Immunofluorescence was performed as previously described in detail [30 (link)]. Cultured cells were grown in chamber slides and fixed in 4% (weight/volume) paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100, blocked with 5% goat serum in phosphate-buffered saline (PBS), and stained using the same protocol as for tissue. We used specific antibodies for pSMAD1 (Millipore, St. Louis, MO), pSMAD2 (ThermoFisher Scientific), HOXD3 (Santa Cruz Biotechnology, Dallas, TX), TGFβ1 and ALK1 (R&D Systems, Minneapolis, MN), and ALK5 (Sigma-Aldrich, St. Louis, MO). The nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich).

Total cell lysates were obtained by lysing the cells in RIPA lysis and extraction buffer (Thermo Fisher Scientific). The protein concentration was determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). The proteins were separated using 12% SDS-PAGE gels (ThermoFisher Scientific) and transferred to nitrocellulose membrane, then incubated with the primary antibodies. The antibodies included phosphorylated (p)-SMAD1/5/8 (Millipore, diluted 1:500), p-SMAD2 (ThermoFisher Scientific, diluted 1:500), HOXD3 and GAPDH (Santa Cruz Biotechnology, diluted 1:200), ALK5 (Sigma-Aldrich, diluted 1:500), TGFβ1 and ALK1 (both from R&D Systems, diluted 1:500). After the membrane was washed three times with Tris-buffered saline with Tween 20 (TBST), it was incubated with secondary antibodies, such as goat anti-rabbit antibodies or goat anti-mouse antibodies (both from Cell Signal Technology, Danvers, MA, diluted 1:1000) or donkey anti-goat antibodies (R&D Systems, diluted 1:5000). The protein expression was normalized to β-actin in each sample.