Ab69498

Antibodies against ALP (ab108337) and OPN (ab69498) were purchased from Abcam (Cambridge, MA, USA). Antibodies against IBSP (5468), ERK1/2 (4370), p-ERK1/2 (4695), MEK1/2 (8727) and p-MEK1/2 (9154) were purchased from Cell Signaling Technology (Danvers, USA). The RUNX2 antibody (sc-390715) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against SPRY4 (22765-1-AP), GAPDH (10494-1-AP) and β-actin (HRP-60008) were purchased from Proteintech (Wuhan, China).
Melatonin was purchased from Sigma-Aldrich (St. Louis, MO, USA). U0126 was purchased from Selleck Chemicals (Houston, TX, USA).

Immunohistochemical (IHC) staining was performed using the Ventana IHC staining system (Ventana, Tucson, AZ). IHC was performed using the primary antibodies against SQS (1:100, GTX104091, Genetex) and OPN (1:1000, ab69498, Abcam). The OPN score was based on the intensity of IHC staining: −, negative; 1+, 0–20% of tumor cell stained; 2+, 20–50% of tumor cell stained; and 3+, >50% of tumor cell stained.

The serum samples of ESCC that were positive response to SPP1 in ELISA were detected by western blotting to confirm the occurrence of immunoreactivity in the sera. Mouse monoclonal anti-SPP1 antibody (1:100 dilution, Abcam, ab69498, USA) was regarded as a positive control. The procedure of western blotting utilized in this study was described in our previous study [26 (link)]. In brief, the recombinant SPP1 protein was electrophoresed by 10% SDS-PAGE and transferred onto a nitrocellulose membrane that was then cut into strips and incubated with selected sera diluted at 1:100, subsequently incubated with mouse anti-human IgG conjugated HRP diluted at 1:5000.

HBMScs were lysed using mammalian protein extraction RIPA reagent (Yeasen). Equivalent amounts (50 μg) of cell protein lysates were electrophoresed on an 10% SDS-polyacrylamide gel and transferred to PVDF membranes (Thermo Fisher Scientific). The membranes were incubated with primary antibodies of RUNX (1:750, ab76956, Abcam, Cambridge, MA, USA), BGLAP (1:1000, ab13421, Abcam), SSP1 (1:750, ab69498, Abcam), and GAPDH (1:1000, 60004-1-Ig, ProteinTech Group, Chicago, IL, USA). After incubating with HRP-associated second antibodies, protein blots were observed using ECL Select Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) and the results were quantified using Image Pro Plus 6.0 (Media Cybernetics).

Tissues were fixed with 4% paraformaldehyde for 48 h, and 3-μm thick tissue sections were prepared. Sections were subsequently deparaffinized and rehydrated using conventional methods. Antigen retrieval was performed with Antigen Unmasking Solution (Vector Laboratories, United States) at 95°C for 10 min. Sections were immersed in 3% hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase activity and were blocked with 10% normal goat serum for 30 min at room temperature. Sections were incubated with primary antibodies against Runx2 (ab76956; 1:200, Abcam, United Kingdom), Osteopontin (OPN; ab69498; 1:200, Abcam, United Kingdom), and osteocalcin (OCN; ab93876; 1:400, Abcam, United Kingdom) at 4°C overnight. Then, sections were incubated with the corresponding biotinylated secondary antibody (Vector Laboratories, United States) for 30 min at room temperature prior to reaction with DAB chromogen (Vector Laboratories, United States). Sections were counterstained with hematoxylin, and micrographs were acquired with a phase contrast light microscope (Leica, Germany).