Anxa6

Western blot analysis was conducted using a previously described protocol.^{39 (link)} We used antibodies against ANXA1, β-catenin, caspase 3, Lrp5, Lrp6, Runx2, Sclerostin, Snail, TGFβ, NFATc1, cathepsin K (all from Cell Signaling, Danvers, MA, USA), DMP1, ANXA6, CXCL5 (all from Abcam, Cambridge, MA, USA), M-CSF, MMP9, OPN, TPM4 (all from Santa Cruz, Dallas, TX, USA), WISP1 (R&D systems, Minneapolis, MN, USA), β-actin (Sigma, Saint Louis, MO, USA), LIMA1, Trail (both from Novus, Centennial, CO, USA), p53, CXCL1 (both from Invitrogen, Carlsbad, California, USA), and DSP (ProteinTech, Rosemont, IL, USA). The expression levels of Sclerostin and Lrp5 in CM were detected by ELISA (My BioSource, San Diego, CA, USA). Proteins isolated from A5 osteocyte CM, Y4 osteocyte CM, and osteoclast control CM (RAW264.7 cells) were analyzed with an HF Hybrid Quadrupole Orbitrap mass spectrometer. Among the 549 identified proteins, 49 proteins had higher expression levels in A5 CM than in Y4 CM and control CM. Among these proteins, 11 (p53; SPARC = osteonectin; TPM1, TPM4 = tropomyosin 1 and 4; ANXA1, ANXA6 = annexin A1 and A6; FMOD = fibromodulin; OGN = osteoglycin; DSP = desmoplakin; AHNAK = desmoyokin; and LIMA1 = LIM domain actin-binding protein 1) were identified as potential tumor suppressors.

Ovarian tissues were embedded in OCT medium for sectioning. Sections (7 μm) were fixed with 4% paraformaldehyde and then permeabilized with 1% Triton X-100. After washing twice with PBS, the sections were incubated with antibodies against S phase related proliferating cell nuclear antigen (PCNA) (Santa Cruz, CA, dilution 1:500), HSP90B1 (CST, dilution 1:200), CALM1, ANXA6, or TPM2 (ABcam, dilution 1:200) for 2 hours. After washing twice with PBS, the sections were incubated with secondary antibodies conjugated to HRP for 1 hour. Finally, the sections were washed 3 times and developed by addition of DAB substrate. Positively stained cells were visualized under a light contrast microscope. Images were obtained using a Nikon Eclipse, TE 2000-U fluorescence microscope.

Formalin-fixed paraffin-embedded tissue sections (8 μm thickness) prepared from xenograft tumors from the AnxA6 down-regulated BT-A6sh5 or the AnxA6-deficient BT-A6A cells were stained as previously described [3 (link)] using the following primary antibodies: RasGRF2 (Abcam, Cambridge, MA), AnxA6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); EGFR (Cell Signaling Technology, Danvers, MA). For quantification of the immunostaining, slides were digitally scanned at the Digital Histology Shared Resource at Vanderbilt University Medical Center. The stained tissue areas were digitally demarcated and the staining intensity analyzed by using the Tissue IA software (Leica Microsystems).