Yt5356

Tissue sections were deparaffinized, dehydrared and then antigen retrieval was done in citric acid buffer (pH 6.0). The tissues were blocked with bovine serum albumin (BSA) for 1 hour before incubation overnight with the anti-IPO7 monoclonal antibody (1:100, sc365231, Santa Cruz) at 4 °C. The sections were then incubated with fluorescent secondary antibodies (1:200, A23210, Abbkine) for 1 hour. Then the tissues were washed 3 times in phosphate buffer saline (PBS), after which they were mounted in fluorescent mounting medium with DAPI (ZSGB-BIO). For cell immunofluorescence, the cells were fixed with 4% PFA at 4 °C, then permeabilized with 0.1% Triton X100 (BioFroxx), blocked by 3% BSA at 37 °C for 1 hour and were incubated with rabbit-anti-RUNX2 (YT5356, Immunoway) and mouse-anti-IPO7 (sc365231, Santa Cruz) antibodies overnight at 4 °C, then washed 3 times with PBS and incubated with fluorescent secondary antibodies for 1 hour (A23220, Abbkine; ANT034, Antgene). The fluorescence images were observed under fluorescence microscopy (Olympus 1 × 83, Japan).

Cells were lysed in NP40 Lysis Buffer with protease inhibitor cocktail (MCE). The cell lysis were analyzed with electrophoresis and then transferred to PVDF membrane (Millipore). The filter was blocked for 1-2 hours by 5% nonfat milk in TBST (Tris buffered saline containing Tween 20) and then incubated with the primary antibodies at 4 °C overnight. The primary antibodies used were: anti-IPO7 monoclonal antibody (sc365231, Santa Cruz), anti-RUNX2 polyclonal antibody (YT5356, Immunoway), anti-KLF4 polyclonal antibody (4038, Cell Signaling technology), anti-Phospho-SMAD1/5 monoclonal antibody (9516, Cell Signaling technology), anti-OSX monoclonal antibody (sc393325, Santa Cruz), anti-DLX3 polyclonal antibody (132613AP, Proteintech), anti-DMP1 polyclonal antibody (ab103203, Abcam), anti-DSP polyclonal antibody (NBP191612, NOVUS), anti-β-ACTIN monoclonal antibody (660091Ig, Proteintech) and anti-PCNA polyclonal antibody (102052AP, Proteintech). After incubation with the corresponding antibodies, the filter was washed 3 times for 5 minutes each with TBST. The filter was then incubated with HRP (horseradish peroxidase) conjugated goat anti-Mouse or anti-Rabbit IgG (Biofly, China) for 1 hour. The filter was washed and developed by a chemiluminescence assay (GE Healthcare). We used ImageJ software for further analysis.