Anti foxf1

Cells and tissue samples were lysed in ice-cold radioimmunoprecipitation assay buffer supplemented with the Protease Inhibitor Cocktail (Sigma-Aldrich). The lysates were resolved in sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk and incubated with anti-PAX8, anti-E-cadherin, anti-vimentin, anti-vascular endothelial growth factor (VEGF), anti-FOXM1, anti-FOXC2, anti-FOXF1, anti-FOXL1, and anti-β-actin (Abcam, Cambridge, MA, USA) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Signals were developed by enhanced chemiluminescence (Merck Millipore, Darmstadt, Germany).

Western blot was performed as previously descripted (Geng et al., 2019 (link)). Antibodies were used: anti-GAPDH (clone 14C10; Cell Signaling Technology), anti-SEMA7A (NBP1-86555; Novus Biologicals), anti-F3 (clone E9M6T; Cell Signaling Technology), anti-ITGA6 (3750S; Cell Signaling Technology), anti-FOXF1 (AF4798; R&D system), anti-CREBRF (ab26262; Abcam), anti-TSC22D1 (NBP2-46238; Novus Biologicals), anti-MXI1 (A12098; ABclonal), anti-KLF9 (clone A-5; Santa Cruz Biotechnology), anti-NFE2L2 (clone D1Z9C; Cell Signaling Technology), anti-HMGA2 (8179s, clone D1A7; Cell Signaling Technology), anti-DPF3 (NBP2-14910; Novus Biologicals), anti-p-HER2 (6942s, clone D66B7; Cell Signaling Technology), and anti-HER2 (2165s, clone 29D8; Cell Signaling Technology). Secondary antibodies were anti-rabbit IgG, HRP-linked Antibody (7074s; Cell Signaling Technology), anti-mouse IgG, HRP-linked antibody (7076s; Cell Signaling Technology), and Peroxidase Donkey Anti-Goat IgG (H + L; 705-036-147; Jackson ImmunoResearch).

Bone tissues were crushed and then lysed in RIPA Lysis Buffer (Invitrogen), BMSCs were also lysed in RIPA Lysis Buffer (Invitrogen), and the extracted proteins were quantified by BCA protein assay reagent (Thermo Fisher Scientific Inc). Proteins (30 μg) were separated by SDS-PAGE before being transferred onto polyvinylidene difluoride membranes. The membranes were blocked by 5% bovine serum albumin, and probed with primary antibodies: anti-MeCP2 and anti-GAPDH (1:2000; Abcam, Cambridge, MA, USA), anti-ALP and anti-RUNX2 (1:2500; Abcam), anti-COL1A1 and anti-OCN (1:3000; Abcam), anti-FOXF1 and anti-Wnt5a (1:3500; Abcam), anti-β-Catenin and anti-APC (1:4000; Abcam). The membrane was washed by phosphate Buffered Saline containing Tween 20 buffer and then incubated with corresponding goat anti-mouse or goat anti-rabbit secondary antibodies (1:4500; Abcam), and the protein strips were visualized by Colorimetric Western blotting Kit (Sigma-Aldrich; St. Louis, MO, USA) according to published work [20 (link)].